Use of recombinant gene delivery vectors for treating or preventing diseases of the eye

ABSTRACT

Gene delivery vectors, such as, for example, recombinant adeno-associated viral vectors, and methods of using such vectors are provided for use in treating or preventing diseases of the eye.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/525,956, filed Mar. 15, 2000, which applicationclaims priority to U.S. Provisional Application No. 60/124,460, filedMar. 15, 1999, and U.S. Provisional Application No. 60/174,984, filedJan. 6, 2000, all of which applications are incorporated by reference intheir entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions andmethods for treating diseases of the eye, and more specifically, to theuse of various gene delivery vectors which direct the expression ofselected gene products suitable for treating or preventing diseases ofthe eye.

BACKGROUND OF THE INVENTION

[0003] Eye diseases represent a significant health problem in the UnitedStates and world-wide. Presently, over 80 million Americans are affectedwith potentially blinding eye disease, with 1.1 million being legallyblind. Twelve million individuals suffer from some degree of visualimpairment that cannot be corrected. The total economic impact of eyedisease is also very significant. In 1981, the estimated economic impactof visual impairment on the U.S. economy was 14 billion per year. By1995, this impact had grown to an estimated 38.4 billion (National EyeInstitute NIH).

[0004] A wide variety of eye diseases can cause visual impairment,including for example, macular degeneration, diabetic retinopathies,inherited retinal degeneration such as retinitis pigmentosa, glaucoma,retinal detachment or injury and retinopathies (whether inherited,induced by surgery, trauma, a toxic compound or agent, or, photically).

[0005] One structure in the eye that can be particularly affected bydisease is the retina. Briefly, the retina, which is found at the backof the eye, is a specialized light-sensitive tissue that containsphotoreceptor cells (rods and cones) and neurons connected to a neuralnetwork for the processing of visual information (see FIG. 10). Thisinformation is sent to the brain for decoding into a visual image.

[0006] The retina depends on cells of the adjacent retinal pigmentepithelium (RPE) for support of its metabolic functions. Photoreceptorsin the retina, perhaps because of their huge energy requirements andhighly differentiated state, are sensitive to a variety of genetic andenvironmental insults. The retina is thus susceptible to an array ofdiseases that result in visual loss or complete blindness.

[0007] Retinitis pigmentosa (RP), which results in the destruction ofphotoreceptor cells, the RPE, and choroid typify inherited retinaldegenerations. This group of debilitating conditions affectsapproximately 100,000 people in the United States.

[0008] A great deal of the progress made in addressing this importantclinical problem has depended on advances in research on photoreceptorcell biology, molecular biology, molecular genetics, and biochemistryover the past two decades. Animal models of hereditary retinal diseasehave been vital in helping unravel the specific genetic and biochemicaldefects that underlie abnormalities in human retinal diseases. It nowseems clear that both genetic and clinical heterogeneity underlie manyhereditary retinal diseases.

[0009] The leading cause of visual loss in the elderly is Age-relatedMacular Degeneration (AMD). The social and economic impact of thisdisease in the United States is increasing. The macula is a structurenear the center of the retina that contains the fovea. This specializedportion of the retina is responsible for the high-resolution vision thatpermits activities such as reading. The loss of central vision in AMD isdevastating. Degenerative changes to the macula (maculopathy) can occurat almost any time in life but are much more prevalent with advancingage. With growth in the aged population, AMD will become a moreprevalent cause of blindness than both diabetic retinopathy and glaucomacombined. Laser treatment has been shown to reduce the risk of extensivemacular scarring from the “wet” or neovascular form of the disease. Theeffects of this treatment are short-lived, however, due to recurrentchoroidal neovascularization. Thus, there are presently no effectivetreatments in clinical use for the vast majority of AMD patients.

[0010] Other diseases of the eye, such as glaucoma, are also majorpublic health problems in the United States. In particular, blindnessfrom glaucoma is believed to impose significant costs annually on theU.S. Government in Social Security benefits, lost tax revenues, andhealthcare expenditures.

[0011] Briefly, glaucoma is not a uniform disease but rather aheterogeneous group of disorders that share a distinct type of opticnerve damage that leads to loss of visual function. The disease ismanifest as a progressive optic neuropathy that, if left untreated,leads to blindness. It is estimated that as many as 3 million Americanshave glaucoma and, of these, as many as 120,000 are blind as a result.Furthermore, it is the number one cause of blindness inAfrican-Americans. Its most prevalent form, primary open-angle glaucoma,can be insidious. This form usually begins in midlife and progressesslowly but relentlessly. If detected early, disease progression canfrequently be arrested or slowed with medical and surgical treatment.

[0012] Glaucoma can involve several tissues in the front and back of theeye. Commonly, but not always, glaucoma begins with a defect in thefront of the eye. Fluid in the anterior portion of the eye, the aqueoushumor, forms a circulatory system that brings nutrients and supplies tovarious tissues. Aqueous humor enters the anterior chamber via theciliary body epithelium (inflow), flows through the anterior segmentbathing the lens, iris, and cornea, and then leaves the eye viaspecialized tissues known as the trabecular meshwork and Schlemm's canalto flow into the venous system. Intraocular pressure is maintainedvis-à-vis a balance between fluid secretion and fluid outflow. Almostall glaucomas are associated with defects that interfere with aqueoushumor outflow and, hence, lead to a rise in intraocular pressure. Theconsequence of this impairment in outflow and elevation in intraocularpressure is that optic nerve function is compromised. The result is adistinctive optic nerve atrophy, which clinically is characterized byexcavation and cupping of the optic nerve, indicative of loss of opticnerve axons.

[0013] Primary open-angle glaucoma is, by convention, characterized byrelatively high intraocular pressures believed to arise from a blockageof the outflow drainage channel or trabecular meshwork in the front ofthe eye. However, another form of primary open-angle glaucoma,normal-tension glaucoma, is characterized by a severe optic neuropathyin the absence of abnormally high intraocular pressure. Patients withnormal-tension glaucoma have pressures within the normal range, albeitoften in the high normal range. Both these forms of primary open-angleglaucoma are considered to be late-onset diseases in that, clinically,the disease first presents itself around midlife or later. However,among African-Americans, the disease may begin earlier than middle age.In contrast, juvenile open-angle glaucoma is a primary glaucoma thataffects children and young adults. Clinically, this rare form ofglaucoma is distinguished from primary open-angle glaucoma not only byits earlier onset but also by the very high intraocular pressureassociated with this disease. Angle-closure glaucoma is a mechanicalform of the disease caused by contact of the iris with the trabecularmeshwork, resulting in blockage of the drainage channels that allowfluid to escape from the eye. This form of glaucoma can be treatedeffectively in the very early stages with laser surgery. Congenital andother developmental glaucomas in children tend to be severe and can bevery challenging to treat successfully. Secondary glaucomas result fromother ocular diseases that impair the outflow of aqueous humor from theeye and include pigmentary glaucoma, pseudoexfoliative glaucoma, andglaucomas resulting from trauma and inflammatory diseases. Blockage ofthe outflow channels by new blood vessels (neovascular glaucoma) canoccur in people with retinal vascular disease, particularly diabeticretinopathy.

[0014] Primary open-angle glaucoma can be insidious. It usually beginsin midlife and progresses slowly but relentlessly. If detected, diseaseprogression can frequently be arrested or slowed with medical andsurgical treatment. However, without treatment, the disease can resultin absolute irreversible blindness. In many cases, even when patientshave received adequate treatment (e.g., drugs to lower intraocularpressure), optic nerve degeneration and loss of vision continuesrelentlessly.

[0015] The present invention provides compositions and methods fortreating and preventing a number of diseases of the eye, including forexample, retinal diseases and degenerations such as RP and AMD, as wellas other diseases such as neovascular disease. The present inventionalso provides other, related advantages.

SUMMARY OF THE INVENTION

[0016] Briefly stated, the present invention provides compositions andmethods for treating, preventing, or, inhibiting diseases of the eye.Within one aspect of the present invention, methods are provided fortreating or preventing diseases of the eye comprising the step ofintraocularly administering a gene delivery vector which directs theexpression of one or more neurotrophic factors, or, anti-angiogenicfactors, such that the disease of the eye is treated or prevented.Within related aspects of the present invention, gene delivery vectorsare provided which direct the expression of one or more neurotrophicfactors such as FGF, as well as gene therapy vectors which direct theexpression of one or more anti-angiogenic factors. Within certainembodiments of the invention, a viral promoter (e.g., CMV) or aninducible promoter (e.g., tet) is utilized to drive the expression ofthe neurotrophic factor.

[0017] Representative examples of gene delivery vectors suitable for usewithin the present invention may be generated from viruses such asretroviruses (e.g., FIV or HIV), herpesviruses, adenoviruses,adeno-associated viruses, and alphaviruses, or from non-viral vectors.

[0018] Utilizing the methods and gene delivery vectors provided herein awide variety of diseases of the eye may be readily treated or prevented,including for example, glaucoma, macular degeneration, diabeticretinopathies, inherited retinal degeneration such as retinitispigmentosa, retinal detachment or injury and retinopathies (whetherinherited, induced by surgery, trauma, a toxic compound or agent, or,photically). Similarly, a wide variety of neurotrophic factors may beutilized (either alone or in combination) within the context of thepresent invention, including for example, NGF, BDNF, CNTF, NT-3, NT-4,FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21.

[0019] Within certain embodiments of the invention, it is preferred thatthe gene delivery vector be utilized to deliver and express ananti-angiogenic factor for the treatment, prevention, or, inhibition ofdiabetic retinopathy, wet AMD, and other neovascular diseases of the eye(e.g., ROP). Within other embodiments it is desirable that the genedelivery vector be utilized to deliver and express a neurotrophic growthfactor to treat, prevent, or, inhibit diseases of the eye, such as, forexample, glaucoma, retinitis pigmentosa, and dry AMD. Within yet otherembodiments, it may be desirable to utilize either a gene deliveryvector which expresses both an anti-angiogenic molecule and aneurotrophic growth factor, or two separate vectors which independentlyexpress such factors, in the treatment, prevention, or inhibition of aneye disease (e.g., for diabetic retinopathy).

[0020] Within further embodiments of the invention, the above-mentionedmethods utilizing gene delivery vectors may be administered along withother methods or therapeutic regimens, including for example,photodynamic therapy (e.g., for wet AMD), laser photocoagulation (e.g.,for diabetic retinopathy and wet AMD), and intraocular pressure reducingdrugs (e.g., for glaucoma).

[0021] Also provided by the present invention are isolated nucleic acidmolecules comprising the sequence of FIG. 2, vectors which contain,and/or express this sequence, and host cells which contain such vectors.

[0022] Within further aspects of the present invention gene deliveryvectors are provided which direct the expression of a neurotrophicfactor, or, an anti-angiogenic factor. As noted above, representativeexamples of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT-4,FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21. Representative examples ofanti-angiogenic factors include soluble Flt-1, soluble Tie-2 receptor,and PEDF. Representative examples of suitable gene delivery vectorsinclude adenovirus, retroviruses (e.g., HIV or FIV-based vectors),alphaviruses, AAV vectors, and naked DNA vectors.

[0023] Within yet other aspects of the invention non-human animal modelsof neovascular diseases of the eye are provided, comprising an animalhaving an angiogenic (i.e., pro-angiogenic) transgene in the eye. Withinvarious embodiments, the neovascularization may be retinal or choroidalneovascularization. Within other embodiments, the animal may be a mouseor rat. As noted herein, a wide variety of angiogenic transgenes may beutilized to generate the non-human animal model, including for example,angiogenic transgenes that encode VEGF and/or an angiopoietin such asangiopoietin-1.

[0024] Also provided are methods of making such non-human animal modelscomprising the general steps of administering to a non-human animal agene delivery vector which directs the expression of an angiogenictransgene. As noted above, a wide variety of gene delivery vectors(e.g., rAV and rAAV) can be utilized, as well as nucleic acid moleculeswhich encode the angiogenic transgene (e.g., nucleic acid moleculesencoding VEGF or angiopoietin). Within certain embodiments, the genedelivery vector can be administered subretinally or intravitreally.Within further embodiments the animal model can be utilized as a modelfor Age-related Macular Degeneration (AMD), diabetic retinopathy, or,retinopathy of prematurity (ROP).

[0025] Also provided are methods for determining the ability of ananti-angiogenic factor to inhibit neovascularization of the eye,comprising the general steps of (a) administering to an animal model asdescribed herein an anti-angiogenic factor, and (b) determining theability of the anti-angiogenic factor to inhibit neovascularization ofthe eye. As noted herein, the anti-angiogenic factor may be administeredby a variety of routes, including for example, topically, subretinally,or, intravitreally. Further, the animal model may be utilized to testthe efficacy of drugs, compounds, or other factors or agents for a widevariety of eye-related neovascular diseases (including AMD and ROP).

[0026] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth hereinwhich describe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a schematic illustration of pKm201bFGF-2.

[0028]FIGS. 2A and 2B are the nucleic acid sequence of pKm201bFGF-2 (SEQI.D. No. ______).

[0029]FIG. 3 is a schematic illustration of pD10-CMV-FGF-5.

[0030]FIG. 4 is a western analysis of FGF-5 rAAV infected 293 cells.

[0031]FIG. 5 is a schematic illustration of pD10-CMV-FGF-5 (sig−).

[0032]FIG. 6 is a western analysis of pD10-CMV-FGF-5 (sig−) transfected293 cells.

[0033]FIG. 7 is a schematic illustration of pD10-CMV-FGF-18.

[0034]FIG. 8 western analysis of 293 cells transfected pD10-CMV-FGF-18.

[0035]FIGS. 9A and 9B are photographs which show that bluo-gal stainingis visible across 40% of a retina transfected with AAV-CMV-lacZ. Allphotoreceptors appear to express lacZ at the injection site, except atthe edge where individual cells are visible.

[0036]FIG. 10 is a schematic illustration which shows the retina withinthe eye, and the organization of cells within the retina.

[0037]FIGS. 11A and 11B are photographs of wild-type and degeneratedS334ter rat retinas. S33 4ter is a rat model for retinitis pigmentosa.

[0038]FIGS. 12A, 12B and 12C are photographs of degenerated S334ter,FGF-2 20 injected S334ter and PBS injected S334ter rat retinas. As canbe seen in these figures, FGF-2 injected into the S334ter rat retinasubstantially slows the progression of disease.

[0039]FIG. 13 is a graph which plots Outer Nuclear Layer (ONL) thicknessfor FGF-2 subretinally injected, PBS subretinally injected, and anuninjected control.

[0040]FIG. 14 is a bar graph which plots ONL thickness at p60.

[0041]FIGS. 15A, 15B and 15C are photographs of FGF-2 expressing cellsstained with an anti-FGF-2 antibody.

[0042]FIGS. 16A and 16B are photographs which show gene delivery tocells in the ganglion cell layer following intraocular injection ofrecombinant rAAV-CMV-lacZ. a) superior quadrant of a retinal flat-mountprocessed for Bluo-Gal staining to visualize AAV-infected infectedneurons. Notice the large number of axons converging at the optic nervehead (asterisk). b) Retinal radial section showing the AAV-mediated LacZgene product in cells of the ganglion cell layer. A large number ofthese cells can be identified as RGCs because of the intense LacZstaining in axons projecting to the optic nerve head (asterisk). RPE:retinal pigment epithelium, PS: photoreceptor segments, ONL: outernuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer,IPL: inner plexiform layer, GCL: ganglion cell layer. Scale bars: a) 0.5mm, b) 50 μm.

[0043]FIG. 17 is a graph which shows the time-course of AAV-mediatedtransgene expression in the ganglion cell layer of the adult rat retina.A recombinant AAV vector (rAAV-CMV-lacZ) was injected into the vitreouschamber of adult rats and 2, 4 and 8 weeks later, Lac-Z positive neuronsin the ganglion cell layer (GCL) were counted in retinal flat-mounts.The values are the mean of 3-4 retinas per time point +standarddeviation (p<0.001).

[0044]FIGS. 18A and 18B are photographs which show the localization ofthe AAV-mediated LacZ gene product in retrogradely labeled RGCs. a)Retinal radial section showing LacZ immunopositive RGCs transduced withAAV (excitation 520-560, barrier 580, emission 580); b) Same sectionexamined under a different fluorescent filter (excitation 355-425,barrier 460, emission 470) to visualize RGCs backlabeled with Fluorogoldfrom the superior colliculus. Notice that the vast majority of LacZimmunopositive neurons are also labeled with Fluorogold. An exception, aLacZ positive cell that is not Fluorogold labeled, is shown (arrow), andcould represent a displaced amacrine cell or RGC that did notincorporate the retrograde tracer. INL: inner nuclear layer, IPL: innerplexiform layer; GCL: ganglion cell layer. Scale bar: 10 μm.

[0045]FIG. 19 is a graph which quantifies Fluorogold- and LacZ-positivecells in the ganglion cell layer following intravitreal injection ofrAAV-CMV-lacZ. The number of Fluorogold-positive cells (FG+) wascompared to the number of cells that expressed both the Fluorogold andLacZ markers (FG+, LacZ+) and the number of cells expressing only thereporter gene (LacZ+). Values represent the mean of 4-5 retinal radialsections per animal (n=4)±standard deviation (S.D.) (p<0.001).

[0046]FIG. 20 is a photograph which shows the localization of theheparan sulfate (HS) proteoglycan receptor, the cellular receptor forAAV, in the intact adult rat retina. Retinal cryosection immunostainedwith a polyclonal antibody against the heparan sulfate (HepSS-1; diluted1:200) followed by biotinylated anti-rabbit Fab fragment,avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame,Calif.). The section was reacted in a solution containing 0.05%diaminobenzidine tetrahydrochloride (DAB) and 0.06% hydrogen peroxide inPB (pH 7.4) for 5 min. Notice the strong labeling in RGCs in theganglion cell layer (GCL). RPE: retinal pigment epithelium, PS:photoreceptor segments, ONL: outer nuclear layer, OPL: outer plexiformlayer, INL: inner nuclear layer, IPL: inner plexiform layer. Scale bar:50 μm.

[0047]FIG. 21 is a schematic illustration of pD10-VEGFuc.

[0048]FIGS. 22A and 22B are the nucleotide sequence of pD10-VEGFUC.

[0049]FIG. 23 is a bar graph which shows pD10-VEGFUC rAAV virusinfection of 293 cells.

[0050]FIG. 24 is a three dimensional bar-graph which shows VEGFsecretion by hfRPE after infection with VEGF AAV.

[0051]FIG. 25 is a three dimensional bar-graph which shows VEGFsecretion by hfRPE after infection with VEGF AV

[0052]FIG. 26 is a three dimensional bar-graph which shows resistance ofhfRPE after infection with VEGF AV.

[0053]FIG. 27 is a schematic illustration of pD10-sFlt-1.

[0054]FIGS. 28A and 28B provide the nucleotide sequence of pD10-sFlt-1.

[0055]FIG. 29 is the nucleotide sequence of FGF-20.

[0056]FIG. 30 is the nucleotide sequence of FGF-21.

[0057]FIG. 31 is a schematic illustration of pD10K-FGF-2Sc.

[0058]FIGS. 32A and 32B are the nucleotide sequence of pD10K-FGF-2Sc.

[0059]FIG. 33 is a graph that compares ONL thickness (um) afterinjection of various vectors into the eye.

[0060]FIG. 34 is a bar graph that shows inhibition of HMVECproliferation by sFlt-1 rAAV.

[0061]FIGS. 35A and 35B are photographic images showing retinal bloodvessels from a live animal, before sacrifice.

[0062]FIGS. 36A, 36B, 36C, and 36D are a series of images showing epoxysections of an eye at various distances from an AAV-VEGF injection site.

[0063]FIGS. 37A, 37B, 37C, and 37D are photographs which showlectin/BrdU double-staining of the rat retina.

[0064]FIGS. 38A and 38B are bar graphs which show sFlt-1 and PEDF rescueof ERGs.

[0065]FIG. 39 is a graph which shows the ERG of a test and control eyeof sFlt-1 treated rats.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0066] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used hereinafter.

[0067] “Gene delivery vector” refers to a construct which is capable ofdelivering, and, within preferred embodiments expressing, one or moregene(s) or sequencers) of interest in a host cell. Representativeexamples of such vectors include viral vectors, nucleic acid expressionvectors, naked DNA, and certain eukaryotic cells (e.g., producer cells).

[0068] “Recombinant adeno-associated virus vector” or “rAAV vector”refers to a gene delivery vector based upon an adeno-associated virus.The rAAV vectors, should contain 5′ and 3′ adeno-associated virusinverted terminal repeats (ITRs), and a transgene or gene of interestoperatively linked to sequences which regulate its expression in atarget cell. Within certain embodiments, the transgene may be operablylinked to a heterologous promoter (such as CMV), or, an induciblepromoter such as (tet). In addition, the rAAV vector may have apolyadenylation sequence.

[0069] “Neurotrophic Factor” or “NT” refers to proteins which areresponsible for the development and maintenance of the nervous system.Representative examples of neurotrophic factors include NGF, BDNF, CNTF,NT-3, NT-4, and Fibroblast Growth Factors.

[0070] “Fibroblast Growth Factor” or “FGF” refers to a family of relatedproteins, the first of which was isolated from the pituitary gland (seeGospodarowicz, D., Nature, 249:123-127, 1974). From this original FGF(designated basic FGF) a family of related proteins, protein muteins,and protein analogs have been identified (see, e.g., U.S. Pat. Nos.4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293,5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226),all of which are generally referred to as Fibroblast Growth Factorswithin the context of the present invention.

[0071] “Anti-anziogenic Factor” refers to a factor or molecule which isable to inhibit the proliferation of vascular growth. A variety ofassays may be utilized to assess the anti-angiogenic activity of a givenmolecule, including for example, the assay provided in Example 15, whichmeasures HMVEC (human dermal microvascular endothelial cell)proliferation. Representative examples of anti-angiogenic factorsinclude for example, Angiostatin, 1,25-Di-hydroxy-vitamn D₃, Endostatin,IGF-1 receptor antagonists, Interferons alpha, beta and gamma,Interferon gamma-inducible protein IP-10, Interleukin 1 alpha and beta,Interleukin 12, 2-Methoxyestradiol, PEDF, Platelet factor 4, Prolactin(16kd fragment), Protamin, Retinoic acid, Thrombospondin-1 and 2, Tissueinhibitor of metalloproteinase-1 and -2, Transforming growth factorbeta, anti-VEGF antibodies (which should be understood to includefragments of antibodies such as a single chain antibodies, Fabfragements, or, CDR regions), soluble Tie-2 receptor, soluble Tie-2receptor, soluble Flt-1 and Tumor necrosis factor-alpha.

[0072] “Diseases of the Eve” refers to a broad class of diseases whereinthe functioning of the eye is affected due to damage or degeneration ofthe photoreceptors; ganglia or optic nerve; or neovascularization.Representative examples of such diseases include macular degeneration,diabetic retinopathies, inherited retinal degeneration such as retinitispigmentosa, glaucoma, retinal detachment or injury and retinopathies(whether inherited, induced by surgery, trauma, a toxic compound oragent, or, photically).

[0073] As noted above, the present invention provides compositions andmethods for treating, preventing, or, inhibiting diseases of the eye,comprising the general step of administering intraocularly a recombinantadeno-associated viral vector which directs the expression of one ormore neurotrophic factors, such that the disease of the eye is treatedor prevented. In order to further an understanding of the invention, amore detailed discussion is provided below regarding (A) gene deliveryvectors; (B) Neurotrophic Factors; (C) Anti-angiogenic factors; and (D)methods of administering the rAAVs in the treatment or prevention ofdiseases of the eye.

[0074] A. Gene Delivery Vectors

[0075] 1. Construction of Retroviral Gene Delivery Vectors

[0076] Within one aspect of the present invention, retroviral genedelivery vectors are provided which are constructed to carry or expressa selected gene(s) or sequence(s) of interest. Briefly, retroviral genedelivery vectors of the present invention may be readily constructedfrom a wide variety of retroviruses, including for example, B, C, and Dtype retroviruses as well as spumaviruses and lentiviruses (see RNATumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).Such retroviruses may be readily obtained from depositories orcollections such as the American Type Culture Collection (“ATCC”;Rockville, Md.), or isolated from known sources using commonly availabletechniques.

[0077] Any of the above retroviruses may be readily utilized in order toassemble or construct retroviral gene delivery vectors given thedisclosure provided herein, and standard recombinant techniques (e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkel, PNAS 82:488, 1985). Inaddition, within certain embodiments of the invention, portions of theretroviral gene delivery vectors may be derived from differentretroviruses. For example, within one embodiment of the invention,retrovirus LTRs may be derived from a Murine Sarcoma Virus, a tRNAbinding site from a Rous Sarcoma Virus, a packaging signal from a MurineLeukemia Virus, and an origin of second strand synthesis from an AvianLeukosis Virus.

[0078] Within one aspect of the present invention, retrovectorconstructs are provided comprising a 5′ LTR, a tRNA binding site, apackaging signal, one or more heterologous sequences, an origin ofsecond strand DNA synthesis and a 3′ LTR, wherein the vector constructlacks gaglpol or env coding sequences.

[0079] Other retroviral gene delivery vectors may likewise be utilizedwithin the context of the present invention, including for example EP0,415,731; WO 90/07936; WO 91/0285, WO 9403622; WO 9325698; WO 9325234;U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, CancerRes. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993;Ram et al., Cancer Res. 53:83-88, 1993; Takamiya et al., J. Neurosci.Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S.Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).

[0080] Packaging cell lines suitable for use with the above describedretrovector constructs may be readily prepared (see U.S. Ser. No.08/240,030, filed May 9, 1994; see also U.S. Ser. No. 07/800,921, filedNov. 27, 1991), and utilized to create producer cell lines (also termedvector cell lines or “VCLs”) for the production of recombinant vectorparticles.

[0081] 2. Recombinant Adeno-Associated Virus Vectors

[0082] As noted above, a variety of rAAV vectors may be utilized todirect the expression of one or more desired neurotrophic factors.Briefly, the rAAV should be comprised of, in order, a 5′adeno-associated virus inverted terminal repeat, a transgene or gene ofinterest operatively linked to a sequence which regulates its expressionin a target cell, and a 3′ adeno-associated virus inverted terminalrepeat. In addition, the rAAV vector may preferably have apolyadenylation sequence.

[0083] Generally, rAAV vectors should have one copy of the AAV ITR ateach end of the transgene or gene of interest, in order to allowreplication, packaging, and efficient integration into cell chromosomes.The ITR consists of nucleotides 1 to 145 at the 5′ end of the AAV DNAgenome, and nucleotides 4681 to 4536 (i.e., the same sequence) at the 3′end of the AAV DNA genome. Preferably, the rAAV vector may also includeat least 10 nucleotides following the end of the ITR (i.e., a portion ofthe “D region”).

[0084] Within preferred embodiments of the invention, the transgenesequence will be of about 2 to 5 kb in length (or alternatively, thetransgene may additionally contain a “stuffer” or “filler” sequence tobring the total size of the nucleic acid sequence between the two ITRsto between 2 and 5 kb). Alternatively, the transgene may be composed ofsame heterologous sequence several times (e.g., two nucleic acidmolecules which encode FGF-2 separated by a ribosome readthrough, oralternatively, by an Internal Ribosome Entry Site or “IRES”), or severaldifferent heterologous sequences (e.g., FGF-2 and FGF-5, separated by aribosome readthrough or an IRES).

[0085] Recombinant AVV vectors of the present invention may be generatedfrom a variety of adeno-associated viruses, including for example,serotypes 1 through 6. For example, ITRs from any AAV serotype areexpected to have similar structures and functions with regard toreplication, integration, excision and transcriptional mechanisms.

[0086] Within certain embodiments of the invention, expression of thetransgene may be accomplished by a separate promoter (e.g., a viralpromoter). Representative examples of suitable promoters in this regardinclude a CMV promoter, RSV promoter, SV40 promoter, or MoMLV promoter.Other promoters that may similarly be utilized within the context of thepresent invention include cell or tissue specific promoters (e.g., arod, cone, or ganglia derived promoter), or inducible promoters.Representative examples of suitable inducible promoters includetetracycline-response promoters (“Tet”, see, e.g., Gossen and Bujard,Proc. Natl. Acad. Sci. USA. 89:5547-5551, 1992; Gossen et al., Science268, 1766-1769, 1995; Baron et al., Nucl. Acids Res. 25:2723-2729, 1997;Blau and Rossi, Proc. Natl. Acad Sci USA. 96:797-799, 1999; Bohl et al.,Blood 92:1512-1517, 1998; and Haberman et al., Gene Therapy 5:1604-1611,1998), the ecdysone system (see e.g., No et al., Proc. Natl. Acad. Sci.USA. 93:3346-3351, 1996), and other regulated promoters or promotersystems (see, e.g., Rivera et al., Nat. Med. 2:1028-1032, 1996;).

[0087] The rAAV vector may also contain additional sequences, forexample from an adenovirus, which assist in effecting a desired functionfor the vector. Such sequences include, for example, those which assistin packaging the rAAV vector in adenovirus-associated virus particles.

[0088] Packaging cell lines suitable for producing adeno-associatedviral vectors may be readily accomplished given readily availabletechniques (see e.g., U.S. Pat. No. 5,872,005).

[0089] Particularly preferred methods for constructing and packagingrAAV vectors are described in more detail below in Examples 1, 2, 3, and4.

[0090] 3. Alphavirus Delivery Vectors

[0091] The present invention also provides a variety of Alphavirusvectors which may function as gene delivery vectors. For example, theSindbis virus is the prototype member of the alphavirus genus of thetogavirus family. The unsegmented genomic RNA (49S RNA) of Sindbis virusis approximately 11,703 nucleotides in length, contains a 5′ cap and a3′ poly-adenylated tail, and displays positive polarity. Infectiousenveloped Sindbis-virus is produced by assembly of the viralnucleocapsid proteins onto the viral genomic RNA in the cytoplasm andbudding through the cell membrane embedded with viral encodedglycoproteins. Entry of virus into cells is by endocytosis throughclatharin coated pits, fusion of the viral membrane with the endosome,release of the nucleocapsid, and uncoating of the viral genome. Duringviral replication the genomic 49S RNA serves as template for synthesisof the complementary negative strand. This negative strand in turnserves as template for genomic RNA and an internally initiated 26Ssubgenomic RNA. The Sindbis viral nonstructural proteins are translatedfrom the genomic RNA while structural proteins are translated from thesubgenomic 26S RNA. All viral genes are expressed as a polyprotein andprocessed into individual proteins by post translational proteolyticcleavage. The packaging sequence resides within the nonstructural codingregion, therefore only the genomic 49S RNA is packaged into virions.

[0092] Several different Sindbis vector systems may be constructed andutilized within the present invention. Representative examples of suchsystems include those described within U.S. Pat. Nos. 5,091,309 and5,217,879, and PCT Publication No. WO 95/07994.

[0093] 4. Other Viral Gene Delivery Vectors

[0094] In addition to retroviral vectors and alphavirus vectors,numerous other viral vectors systems may also be utilized as a genedelivery vector. Representative examples of such gene delivery vectorsinclude viruses such as pox viruses, such as canary pox virus orvaccinia virus (Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner etal., Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); influenzavirus (Luytjes et al., Cell 59:1107-1113, 1989; McMicheal et al., N.Eng. J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978);herpes (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S. Pat. No.5,288,641); HIV (Poznansky, J. Virol. 65:532-536, 1991); measles (EP 0440,219); Semliki Forest Virus, and coronavirus, as well as other viralsystems (e.g., EP 0,440,219; WO 92/06693; U.S. Pat. No. 5,166,057). Inaddition, viral carriers may be homologous, non-pathogenic(defective),replication competent virus (e.g., Overbaugh et al., Science239:906-910,1988), and nevertheless induce cellular immune responses,including CTL.

[0095] 5. Non-viral gene delivery vectors

[0096] In addition to the above viral-based vectors, numerous non-viralgene t delivery vectors may likewise be utilized within the context ofthe present invention. Representative examples of such gene deliveryvectors include direct delivery of nucleic acid expression vectors,naked DNA alone (WO 90/11092), polycation condensed DNA linked orunlinked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3:147-154,1992), DNA ligand linked to a ligand with or without one of the highaffinity pairs described above (Wu et al., J. of Biol. Chem264:16985-16987, 1989), nucleic acid containing liposomes (e.g., WO95/24929 and WO 95/12387) and certain eukaryotic cells (e.g., producercells—see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No.07/800,921).

[0097] B. Neurotrophic Factors

[0098] As noted above, the term neurotrophic factor refers to proteinswhich are responsible for the development and maintenance of the nervoussystem. Representative examples of neurotrophic factors include NGF,BDNF, CNTF, NT-3, NT-4, and Fibroblast Growth Factors.

[0099] Fibroblast Growth Factor refers to a family of related proteins,the first of which was isolated from the pituitary gland (seeGospodarowicz, D., Nature, 249:123-127, 1974). From this original FGF(designated basic FGF) a family of related proteins, protein muteins,and protein analogs have been identified (see, e.g., U.S. Pat. Nos.4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293,5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226;see generally Baird and Gospodarowicz, D. Ann N. Y Acad. Sci. 638:1,1991. The first two members of the family to be identified were acidicfibroblast growth factor (aFGF/FGF-1) and basic fibroblast growth factor(bFGF/FGF-2). Additional members of the FGF family include:i-nt-2/FGF-3, (Smith et al., EMBO J. 7: 1013, 1988); FGF-4 (Delli-Boviet al., Cell 50: 729, 1987); FGF-6 (Marics et al., Oncogene 4: 335(1989); keratinocyte growth factor/FGF-7, (Finch et al., Science 245:752, 1989); FGF-8 (Tanaka et al., Proc. Natl. Acad Sci. USA 89: 8928,1992); and FGF-9 (Miyamoto et al., Mol. Cell Biol. 13: 4251, 1993).

[0100] FGF-5 was originally isolated as an oncogene. See Goldfarb et al.U.S. Pat. Nos. 5,155,217 and 5,238,916, Zhan et al. “Human OncogeneDetected by a Defined Medium Culture Assay” (Oncogene 1:369-376, 1987),Zhan et al. “The Human FGF-5 Oncogene Encodes a Novel Protein Related toFibroblastic Growth Factors” (Molecular and Cellular Biology8:3487-3495, 1988), and Bates et al. “Biosynthesis of Human FibroblastGrowth Factor 5”: (Molecular and Cellular Biology 11:1840-1845, 1991).

[0101] Other FGFs include those disclosed in U.S. Pat. Nos. 4,444,760,5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170,5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226. 5,852,177,and 5,872,226, as well as FGF-20 (U.S. Provisional Application No.60/161,162) and FGF-21 (U.S. Provisional Application No. 60/166,540).

[0102] C. Anti-Angiogenic Factors

[0103] A wide variety of anti-angiogenic factors may also be expressedfrom the gene delivery vectors of the present invention, including forexample, Angiostatin (O'Reilly et al., Cell 79:315-328, 1994; O'Reillyet al., Nat. Med. 2:689-92, 1996; Sim et al., Cancer Res. 57:1329-34,1997), 1,25-Di-hydroxy-vitamn D₃ (Shibuya et al., Oncogene 5:519-24,1990; Oikawa et al., Eur. J Pharmacol. 178:247-50, 1990; and 182:616,1990), Endostatin (O'Reilly et al., Cell 88:277-85, 1997), Interferonsalpha and beta (Sidky et al., Cancer Res. 47:5155-61, 1987; Singh etal., Proc. Natl. Acad. Sci. USA 92:4562-6, 1995), Interferon gamma(Friesel et al., J. Cell. Biol. 104:689-96, 1987), IGF-1 receptorantagonists, Interferon gamma-inducible protein IP-10 (Arenberg et al.,J. Exp. Med. 1996;184:981-92; Strieter et al., J. Leukoc. Biol.1995;57:752-62; Angiolillo et al., J. Exp. Med. 182:155-62, 1995),Interleukin 1alpha and beta (Cozzolino et al., Proc. Natl. Acad. Sci.USA 87:6487-91, 1990), Interleukin 12 (Kerbel and Hawley, J. Natl CancerInst. 87:557-9, 1995; Majewski et al., J. Invest. Dermatol 106:1114-8,1996; Voest et al., J. Natl Cancer Inst. 87:581-6, 1995),2-Methoxyestradiol (Fotsis et al., Nature 368:237-9, 1994), Plateletfactor 4 (Taylor and Folkman, Nature 297:307-12, 1982; Gengrinovitch etal., J. Biol. Chem 270:15059-65, 1995), Prolactin (16kd fragment) (Clappet al., Endocrinology 133:1292-9, 1993; Ferrara, Endocrinology129:896-900, 1991), Protamin, Retinoic acid (Lingen et al., Lab. Invest74:476-83, 1996), Thrombospondin-1 and 2 (Lawler, Blood, 67:1197-209,1986; Raugi and Lovett, Am. J. Pathol 129:364-72, 1987; Volpert et al.,Biochem. Biophys. Res. Commun 217:326-32, 1995), Tissue inhibitor ofmetalloproteinase-1 and -2 (Moses and Langer, J. Cell Biochem 47:230-5,1991; Ray and Stetler-Stevenson, Eur. Respir. J. 7:2062-72, 1994),Transforming growth factor beta (RayChaudhury, J. Cell. Biochem47:224-9, 1991; Roberts et al., Proc. Natl Acad. Sci USA 83:4167-71,1986), and Tumor necrosis factor—alpha (Frater-Schroeder et al., Proc.Natl. Acad. Sci. USSA 84:5277-81, 1987; Leibovich et al., Nature329:630-2, 1987).

[0104] Other anti-angiogenic factors that can be utilized within thecontext of the present invention include VEGF antagonists such assoluble Flt-1 (Kendall and Thomas, PNAS 90: 10705, 1993), pigmentepithelium-derived factor or “PEDF” (Dawson et al., Science 285:245,1999), and Ang-1 antagonists such as soluble Tie-2 receptor (Thurston etal., Science 286:2511, 1999; see also, generally Aiello et al., PNAS92:10457, 1995; Robinson et al., PNAS 93:4851, 1996; Seo et al., Am. J.Pathol. 154:1743, 1999).

[0105] The ability of a given molecule to be “anti-angiogenic” can bereadily assessed utilizing a variety of assays, including for example,the HMVEC assay provided in Example 15.

[0106] D. Method for Treating and/or Preventing Diseases of the Eye, andPharmaceutical Compositions

[0107] As noted above, the present invention provides methods whichgenerally comprise the step of intraocularly administering a genedelivery vector which directs the expression of one or more neurotrophicfactor to the eye, or an anti-angiogenic factor to the eye in order totreat, prevent, or inhibit the progression of an eye disease. Asutilized herein, it should be understood that the terms “treated,prevented, or, inhibited” refers to the alteration of a disease courseor progress in a statistically significant manner. Determination ofwhether a disease course has been altered may be readily assessed in avariety of model systems, discussed in more detail below, which analyzethe ability of a gene delivery vector to delay, prevent or rescuephotoreceptors, as well as other retinal cells, from cell death.

[0108] 1. Diseases of the Eye

[0109] A wide variety of diseases of the eye may be treated given theteachings provided herein. For example, within one embodiment of theinvention gene delivery vectors are administered to a patientintraocularly in order to treat or prevent macular degeneration.Briefly, the leading cause of visual loss in the elderly is maculardegeneration (MD), which has an increasingly important social andeconomic impact in the United States. As the size of the elderlypopulation increases in this country, age related macular degeneration(AMD) will become a more prevalent cause of blindness than both diabeticretinopathy and glaucoma combined. Although laser treatment has beenshown to reduce the risk of extensive macular scarring from the “wet” orneovascular form of the disease, there are currently no effectivetreatments for the vast majority of patients with MD.

[0110] Within another embodiment, gene delivery vectors can beadministered to a patient intraocularly in order to treat or prevent aninherited retinal degeneration. One of the most common inherited retinaldegenerations is retinitis pigmentosa (RP), which results in thedestruction of photoreceptor cells, and the RPE. Other inheritedconditions include Bardet-Biedl syndrome (autosomal recessive);Congenital amaurosis (autosomal recessive); Cone or cone-rod dystrophy(autosomal dominant and X-linked forms); Congenital stationary nightblindness (autosomal dominant, autosomal recessive and X-linked forms);Macular degeneration (autosomal dominant and autosomal recessive forms);Optic atrophy, autosomal dominant and X-linked forms); Retinitispigmentosa (autosomal dominant, autosomal recessive and X-linked forms);Syndromic or systemic retinopathy (autosomal dominant, autosomalrecessive and X-linked forms); and Usher syndrome (autosomal recessive).This group of debilitating conditions affects approximately 100,000people in the United States alone.

[0111] As noted above, within other aspects of the invention, genedelivery vectors which direct the expression of a neurotrophic growthfactor can be administered to a patient intraocularly in order to treator prevent glaucoma. Briefly, glaucoma is not a uniform disease butrather a heterogeneous group of disorders that share a distinct type ofoptic nerve damage that leads to loss of visual function. The disease ismanifest as a progressive optic neuropathy that, if left untreated leadsto blindness. It is estimated that as many as 3 million Americans haveglaucoma and, of these, as many as 120,000 are blind as a result.Furthermore, it is the number one cause of blindness inAfrican-Americans. Its most prevalent form, primary open-angle glaucoma,can be insidious. This form usually begins in midlife and progressesslowly but relentlessly. If detected early, disease progression canfrequently be arrested or slowed with medical and surgical treatment.Representative factors that may be expressed from the vectors of thepresent invention to treat glaucoma include neurotrophic growth factorssuch as FGF-2, 5, 18, 20, and, 21.

[0112] Within yet other embodiments gene delivery vectors can beadministered to a patient intraocularly in order to treat or preventinjuries to the retina, including retinal detachment, photicretinopathies, surgery-induced retinopathies, toxic retinopathies,retinopathies due to trauma or penetrating lesions of the eye.

[0113] As noted above, the present invention also provides methods oftreating, preventing, or, inhibiting neovascular disease of the eye,comprising the step of administering to a patient a gene delivery vectorwhich directs the expression of an anti-angiogenic factor.Representative examples of neovascular diseases include diabeticretinopathy, AMD (wet form), and retinopathy of prematurity. Briefly,choroidal neovascularization is a hallmark of exudative or wetAge-related Macular Degeneration (AMD), the leading cause of blindnessin the elderly population. Retinal neovascularization occurs in diseasessuch as diabetic retinapathy and retinopathy of prematurity (ROP), themost common cause of blindness in the young.

[0114] Particularly preferred vectors for the treatment, prevention, or,inhibition of neovascular diseases of the eye direct the expression ofan anti-angiogenic factor such as, for example, soluble tie-2 receptoror soluble Flt-1.

[0115] 2. Animal Models

[0116] In order to assess the ability of a selected gene therapy vectorto be effective for treating diseases of the eye which involveneovascularization, a novel model for neovascularization (eitherchoroidal or subretinal) can be generated by subretinal injection of arecombinant virus (e.g., rAV or rAAV) containing an angiogenic transgenesuch as VEGF and/or angiopoietin. Within certain embodiments, anangiogenic transgene such as angiopoietin-1 can be used in combinationwith another factor such as VEGF, in order to generateneovascularization. The extent and duration of neovascularizationinduced by the gene delivery vectors containing an angiogenic transgenesuch as VEGF can be determined using fundus photography, fluoresceinangiography and histochemistry.

[0117] To assess the ability of anti-angiogenic molecules to preventneovascularization in the model described above, a D10-sFlt-1 rAAV (orother gene delivery vector which directs the expression of ananti-angiogenic factor) is intraocularly injected, either by subretinalor intravitreal routes of injection. Generally, subretinal injection ofthe gene delivery vector may be utilized to achieve delivery to both thechoroidal and inner retinal vasculature. Intravitreal injection can beutilized to infect Muller cells and retinal ganglion cells (RGCs), whichdeliver anti-angiogenic protein to the retinal vasculature. Muller cellsspan the retina and would secrete the therapeutic protein into thesubretinal space.

[0118] Such injections may be accomplished either prior to, simultaneouswith, or subsequent to administration of an angiogenic factor or genedelivery vector which expresses an angiogenic factor. After anappropriate time interval, inhibition of neovascularization can bedetermined using fundus photography, fluorescein angiography and/orhistochemistry.

[0119] While there are many animal models of retinal neovascularizationsuch as oxygen-induced ischemic retinopathy (Aiello et al., PNAS 93:4881, 1996.) and the VEGF transgenic mouse (Okamoto et al., Am. J.Pathol. 151: 281, 1997), there are fewer models of choroidalneovascularization (e.g., laser photocoagulation as described by Murataet al., IOVS 39: 2474, 1998). Subretinal neovascularization from theretinal rather than choroidal blood supply is also observed in VEGFtransgenic animals (Okamoto et al., Am. J. Pathol. 151: 281, 1997).Hypoxic stimulation of VEGF expression is known to correlate withneovascularization in human ocular disease.

[0120] The pathologic hallmark of glaucomatous optic neuropathy is theselective death of retinal ganglion cells (RGCs) (Nickells, R. W., J.Glaucoma 5:345-356. 1996; Levin, L. A. and Louhab, A., Arch. Ophthalmol.114:488-491, 1996.; Kerrigan, L. A., Zack, D. J., Quigley, H. A., Smith,S. D. and Pease, M. E., Arch. Ophthalmol. 115:1031-1035, 1997). Recentstudies indicate that RGCs die with characteristics of apoptosis afterinjury to the axons of adult RGCs such as axotomy of the optic nerve(ON), and in glaucoma and anterior ischemic optic neuropathy in humans(Nickells, 1996). Thus, damage to the optic nerve by axotomy is used bymany researchers as a model for selective apoptotic cell death of adultRGCs.

[0121] The loss of RGCs caused by ON transection in adult mammals variesfrom 50% to more than 90% depending on the techniques used to identifyRGCs, the proximity of the lesion to the eye, and the age and species ofthe animal. For example, in a study in adult rats, in which retrogradelytransported tracers were used to distinguish RGCs from displacedamacrine cells (Villegas-Perez, M. P., Vidal-Sanz, M., Bray, G. M. andAguayo, A. J., J. Neurosci. 8:265-280, 1988). ON transection near theeye (0.5-1 mm) leads to the loss of more than 90% of the RGCs by 2weeks. In contrast, in adult animals in which the ON was cut nearly 10mm from the eye, 54% of RGCs survived by 3 months (Richardson, P. M.,Issa, V. M. K. and Shemie, S., J. Neurocytol. 11:949-966, 1982.).

[0122] Briefly, the posterior pole of the left eye and the origin of theoptic nerve (ON) are exposed through a superior temporal intraorbitalapproach. A longitudinal excision of the ON dural sheath is performed.The ON is then gently separated from the dorsal aspect of this sheathand completely transected within the orbit, within 1 mm of the opticdisc. Care is taken to avoid damage to the ophthalmic artery, which islocated on the inferomedial dural sheath of the ON.

[0123] RGC survival and death following gene delivery can also beexamined using two alternative models of ON injury: 1) ON crush; and (2)increased intraocular pressure. In the first model the ON is exposed,and then clamped at a distance of about one millimeter from theposterior pole using a pair of calibrated forceps as previouslydescribed (Li et al., Invest. Ophthalmol. Vis. Sci. 40:1004, 1999). Inthe second model, chronic moderately elevated intraocular pressure canbe produced unilaterally by cauterization of three episcleral vessels asdescribed by Neufeld et al. in PNAS 17:9944, 1999).

[0124] A variety of animal models can be utilized for photoreceptordegeneration, including the RCS rat model, P23H transgenic rat model,the rd mouse, and the S334ter transgenic rat model.

[0125] Briefly, in the S334ter transgenic rat model, a mutation occursresulting in the truncation of the C-terminal 15 amino acid residues ofrhodopsin (a seven-transmembrane protein found in photoreceptor outersegments, which acts as a photopigment). The S334ter mutation is similarto rhodopsin mutations found in a subset of patients with retinitispigmentosa (RP). RP is a heterogeneous group of inherited retinaldisorders in which individuals experience varying rates of vision lossdue to photoreceptor degeneration. IN many RP patients, photoreceptorcell death progresses to blindness. Transgenic S334ter rats are bornwith normal number of photoreceptors. The mutant rhodopsin gene beginsexpression at postnatal day 5 in the rat, and photoreceptor cell deathbegins at postnatal day 10-15. In transgenic line S334ter-3,approximately 70% of the outer nuclear layer has degenerated by day 60in the absence of any therapeutic intervention. The retinal degenerationin this model is consistent from animal to animal and follows apredictable and reproducible rate. This provides an assay fortherapeutic effect by morphological examination of the thickness of thephotoreceptor nuclear layer and comparison of the treated eye to theuntreated (contralateral) eye in the same individual animal.

[0126] S334ter rats are utilized as a model for RP as follows. Briefly,S334ter transgenic rats are euthanized by overdose of carbon dioxideinhalation and immediately perfused intracardially with a mixture ofmixed aldehydes (2% formaldehyde and 2.5% glutaraldehyde). Eyes areremoved and embedded in epoxy resin, and 1 μm thick histologicalsections are made along the vertical meridian. Tissue sections arealigned so that the ROS and Müller cell processes crossing the innerplexiform layer are continuous throughout the plane of section to assurethat the sections are not oblique, and the thickness of the ONL andlengths of RIS and ROS are measured. These retinal thicknessmeasurements are plotted and establish the baseline retinal degenerationrates for the animal model. The assessment of retinal thickness is asfollows: briefly, 54 measurements of each layer or structure were madeat set points around the entire retinal section. These data were eitheraveraged to provide a single value for the retina, or plotted as adistribution of thickness or length across the retina. The greatest 3contiguous values for ONL thickness in each retina is also compared inorder to determine if any region of retina (e.g., nearest the injectionsite) showed proportionally greater rescue; although most of thesevalues were slightly greater than the overall mean of all 54 values,they were no different from control values than the overall mean. Thus,the overall mean was used in the data cited, since it was based on amuch larger number of measurements.

[0127] One particularly preferred line of transgenic rats, TgN(s3 34ter)line 4 (abbreviated s334ter 4) can be utilized for in vivo experiments.Briefly, in this rat model expression of the mutated opsin transgenebegins at postnatal day P5 in these rats, leading to a gradual death ofphotoreceptor cells. These rats develop an anatomically normal retina upto P15, with the exception of a slightly increased number of pyknoticphotoreceptor nuclei in the outer nuclear layer (ONL) than innon-transgenic control rats. In this animal model , the rate ofphotoreceptor cell death is approximately linear until P60, resulting inloss of 40-60% of the photoreceptors. After P60, the rate of cell lossdecreases, until by one year the retinas have less than a single row ofphotoreceptor nuclei remaining.

[0128] 3. Methods of Administration

[0129] Gene delivery vectors of the present invention may beadministered intraocularly to a variety of locations depending on thetype of disease to be treated, prevented, or, inhibited, and the extentof disease. Examples of suitable locations include the retina (e.g., forretinal diseases), the vitreous, or other locations in or adjacent tothe eye.

[0130] Briefly, the human retina is organized in a fairly exact mosaic.In the fovea, the mosaic is a hexagonal packing of cones. Outside thefovea, the rods break up the close hexagonal packing of the cones butstill allow an organized architecture with cones rather evenly spacedsurrounded by rings of rods. Thus in terms of densities of the differentphotoreceptor populations in the human retina, it is clear that the conedensity is highest in the foveal pit and falls rapidly outside the foveato a fairly even density into the peripheral retina (see Osterberg, G.(1935) Topography of the layer of rods and cones in the human retina.Acta Ophthal. (suppl.) 6, 1-103; see also Curcio, C. A., Sloan, K. R.,Packer, O., Hendrickson, A. E. and Kalina, R. E. (1987) Distribution ofcones in human and monkey retina: individual variability and radialasymmetry. Science 236, 579-582).

[0131] Access to desired portions of the retina, or to other parts ofthe eye may be readily accomplished by one of skill in the art (see,generally Medical and Surgical Retina: Advances, Controversies, andManagement, Hilel Lewis, Stephen J. Ryan, Eds., medical illustrator,Timothy C. Hengst. St. Louis: Mosby, c1994. xix, 534; see also Retina,Stephen J. Ryan, editor in chief,. 2nd ed., St. Louis, Mo.: Mosby,c1994. 3 v. (xxix, 2559 p.)

[0132] The amount of the specific viral vector applied to the retina isuniformly quite small as the eye is a relatively contained structure andthe agent is injected directly into it. The amount of vector that needsto be injected is determined by the intraocular location of the chosencells targeted for treatment. The cell type to be transduced will bedetermined by the particular disease entity that is to be treated.

[0133] For example, a single 20-microliter volume (of 10¹³ physicalparticle titer/ml rAAV) may be used in a subretinal injection to treatthe macula and fovea. A larger injection of 50 to 100 microliters may beused to deliver the rAAV to a substantial fraction of the retinal area,perhaps to the entire retina depending upon the extent of lateral spreadof the particles.

[0134] A 100-ul injection will provide several million active rAAVparticles into the subretinal space. This calculation is based upon atiter of 10¹³ physical particles per milliliter. Of this titer, it isestimated that {fraction (1/1000)} to {fraction (1/10,000)} of the AAVparticles are infectious. The retinal anatomy constrains the injectionvolume possible in the subretinal space (SRS). Assuming an injectionmaximum of 100 microliters, this would provide an infectious titer of10⁸ to 10⁹ rAAV in the SRS. This would have the potential of infectingall of the 150×10 6 photoreceptors in the entire human retina with asingle injection.

[0135] Smaller injection volumes focally applied to the fovea or maculamay adequately transfect the entire region affected by the disease inthe case of macular degeneration or other regional retinopathies.

[0136] Gene delivery vectors can alternately be delivered to the eye byintraocular injection into the vitreous. In this application, theprimary target cells to be transduced are the retinal ganglion cells,which are the retinal cells primarily affected in glaucoma. In thisapplication, the injection volume of the gene delivery vector could besubstantially larger, as the volume is not constrained by the anatomy ofthe subretinal space. Acceptable dosages in this instance can range from25 ul to 1000 ul.

[0137] 4. Assays

[0138] A wide variety of assays may be utilized in order to determineappropriate dosages for administration, or to assess the ability of agene delivery vector to treat or prevent a particular disease. Certainof these assays are discussed in more detail below.

[0139] a. Electroretinographic Analysis

[0140] Electroretinographic analysis can be utilized to assess theeffect of gene delivery administration into the retina. Briefly, ratsare dark adapted overnight and then in dim red light, then anesthetizedwith intramuscular injections of xylazine (13 mg/kg) and ketamine (87mg/kg). Full-field scotopic ERGs are elicited with 10-μsec flashes ofwhite light and responses were recorded using a UTAS-E 2000 VisualElectrodiagnostic System (LKC Technologies, Inc., Gaithersburg, Md.).The corneas of the rats are the anesthetized with a drop of 0.5%proparacaine hydrochloride, and the pupils dilated with 1% atropine and2.5% phenylephrine hydrochloride. Small contact lenses with gold wireloops are placed on both corneas with a drop of 2.5% methylcellulose tomaintain corneal hydration. A silver wire reference electrode is placedsubcutaneously between the eyes and a ground electrode is placedsubcutaneously in the hind leg. Stimuli are presented at intensities of−1.1, 0.9 and 1.9 log cd m⁻² at 10-second, 30-second and 1-minuteintervals, respectively. Responses are amplified at a gain of 4,000,filtered between 0.3 to 500 Hz and digitized at a rate of 2,000 Hz on 2channels. Three responses are averaged at each intensity. The a-wavesare measured from the baseline to the peak in the cornea-negativedirection, and b-waves are measured from the cornea-negative peak to themajor cornea-positive peak. For quantitative comparison of differencesbetween the two eyes of rats, the values from all the stimulusintensities are averaged for a given animal.

[0141] b. Retinal Tissue Analysis

[0142] As described in more detail above and below, retinal tissueanalysis can also be utilized to assess the effect of gene deliveryadministration into the retina.

[0143] 5. Pharmaceutical Compositions

[0144] Gene delivery vectors may be prepared as a pharmaceutical productsuitable for direct administration. Within preferred embodiments, thevector should be admixed with a pharmaceutically acceptable carrier forintraocular administration. Examples of suitable carriers are saline orphosphate buffered saline.

Deposit Information

[0145] The following material was deposited with the American TypeCulture Collection: Name Deposit Date Accession No. PKm201bFGF-2 3/11/99#207160 PD10-Kan-FGF-2-Sc

[0146] The above material was deposited by Chiron Corporation with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, telephone 703-365-2700. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for purposes of PatentProcedure. The deposit will be maintained for a period of 30 yearsfollowing issuance of this patent, or for the enforceable life of thepatent, whichever is greater. Upon issuance of the patent, the depositswill be available to the public from the ATCC without restriction.

[0147] This deposit is provided merely as a convenience to those ofskill in the art, and is not an admission that a deposit is requiredunder 35 U.S.C. §112. The nucleic acid sequence of this deposit, as wellas the amino acid sequence of the polypeptide encoded thereby, areincorporated herein by reference and should be referred to in the eventof an error in the sequence described herein. A license may be requiredto make, use, or sell the deposited materials, and no such license isgranted hereby.

[0148] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1 Construction of a rAAV Vector Expressing FGF-2

[0149] pKm201CMV is an AAV cloning vector in which an expressioncassette, consisting of a CMV immediate early promoter/enhancer and abovine growth hormone (BGH) polyadenylation site, is flanked by invertedterminal repeat (ITR) sequences from AAV-2. Briefly, pKm201CMV wasderived from pKm201, a modified AAV vector plasmid in which theampicillin resistance gene of pEMBL-AAV-ITR (see Srivastava, (1989)Proc. Natl. Acad. Sci. USA 86:8078-8082) had been replaced with the genefor kanamycin resistance. The expression cassette from pCMVlink, aderivative of pCMV6c (see Chapman, Nucleic Acids Res. 19:193-198 (1991))in which the BGH poly A site has been substituted for the SV40terminator, was inserted between the ITRs of pKm201 to generate pKm201CMV.

[0150] pKm201 bFGF-2 was constructed by cloning the following, in order,into the multiple cloning site of pKm201 CMV: the encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES), the bovine FGF-2 cDNA,and the human growth hormone polyadenylation sequence. The cDNA forFGF-2 has two mutations that change amino acid 121 from serine tothreonine and amino acid 137 from proline to serine. The DNA sequence ofpKm201bFGF-2 is shown in FIG. 2 and the plasmid has been deposited withthe American Type Culture Collection (ATCC).

[0151] rAAV vector particles were produced by a triple transfectionprotocol (Nucleic Acids Res. 24:596-601, 1996; J. Exp. Med.179:1867-1875, 1994). Briefly, human embryonic kidney 293 cells, grownto 50% confluence in a 10 layer Nunclon cell factory (Nalge Nunc, Int.,Naperville, Ill.), were co-transfected with 400 μg of helper plasmidpKSrep/cap (Hum. Gene Ther. 9:477-485, 1998) 400 μg of vector plasmid,and 800 μg of adenovirus plasmid pBHG10 (Microbix Biosystems, Inc.,Toronto, Ontario) using the calcium phosphate co-precipitation method.Forty-eight hours after co-transfection, media was replaced withIMDM+10% FB S containing adenovirus type 5 dl312 at a multiplicity ofinfection (MOI) of 2. Seventy-two hours after infection cells wereharvested and resuspended in HEPES buffer (200 ml total) and lysed bythree cycles of freezing and thawing. Cell debris was removed bycentrifugation at 12,000× g for 20 min. Packaged rAAV was purified fromadenovirus by two rounds of cesium chloride equilibrium density gradientcentrifugation. Residual adenovirus contamination was inactivated byheating at 56° C. for 45 min. Though three plasmids were used in theproduction of rAAV vector in this example, it is possible to combine therAAV vector construct and the AAV helper gene construct on one plasmid.This would allow rAAV to be produced by transfecting 293 cells with twoplasmids. Alternatively, one could add the adenovirus helper genes tothis plasmid to make a single plasmid containing all that is required tomake rAAV particles.

[0152] To estimate total number of rAAV particles, the virus stock wastreated with DNAse I, and encapsidated DNA was extracted withphenol-chloroform, and precipitated with ethanol. DNA dot blot analysisagainst a known standard was used to determine titer (Blood76:1997-2000, 1990).

[0153] To assay for adenovirus contamination, 293 cells were infectedwith 10 μl of purified rAAV stock and followed for any signs ofcytopathic effect. All stocks were negative for adenovirus contamination(level of detection greater than or equal to 100 PFU/ml).

[0154] To assay for wild type AAV, 293 cells were co-infected withserial dilutions of rAAV stocks and adenovirus dl312 at a MOI of 2.Three days later the cells were harvested, lysed by three cycles offreezing/thawing, and centrifuged to remove cell debris. The supernatantwas heat inactivated (56° C. for 10 min) and fresh plates of 293 cells(6×10⁶) were infected in the presence of adenovirus dl312 at a MOI of 2.Forty-eight hours after infection, low molecular-weight DNA was isolated(J Mol. Biol. 26:365-369, 1967) subjected to agarose gelelectrophoresis, and transferred to a nylon membrane. The membrane washybridized with a biotinylated oligonucleotide probe specific for theAAV capsid region. The wild type AAV titer was defined as the highestdilution of rAAV vector stock demonstrating a positive hybridizationsignal. The rAAV preparations contained less than 1 wild type AAV genomeper 109 rAAV genomes.

Example 2 Infection of Cells with RAAV-CMV-FGF-2 Results in theExpression of FGF-2

[0155] 293 cells were plated the day before infection at 5×10⁵cells/well in 6-well plates and were infected with rAAV-CMV-bFGF-2virus, prepared as described above in Example 1, at differentmultiplicities of infection (MOI) with and without etoposide (3μM).Etoposide is a topoisomerase inhibitor which has been shown to increasetransduction efficiency of rAAV vectors (Proc. Natl. Acad. Sci. USA,92:5719-5723, 1995). Forty-eight hours after infection, culturesupernatant was collected and cells were lysed in 0.5 mL 1× lysis buffer(100 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 0.5% NP40, and 0.5%deoxycholate). FGF-2 in the supernatants and lysates was assayed byELISA (cat. #DFB00, R & D Systems, Minneapolis, Minn.) followingmanufacturer's instructions. The results are shown below in Table 1.TABLE 1 FGF-2 PRODUCTiON IN 293 CELLS FOLLOWING INFECTION WITHRAAV-FGF-2 sample infection MOI Etoposide FGF2 protein Culture MediumSupernatant (1.5 ml) 1 0 − <5 pg/ml 2 2 × 10⁵ − <5 pg/ml 3 2 × 10⁴ − <5pg/ml 4 2 × 10³ − <5 pg/ml 1 0 + <5 pg/ml 2 2 × 10⁵ + 106 pg/ml ≈ 300pg/24 h/ 10⁶ cells 3 2 × 10⁴ + <5 pg/ml 4 2 × 10³ + <5 pg/ml Cell Lysate(0.5 ml) 1 0 − 8.95 ng/ml 2 2 × 10⁵ − 114. ng/ml 3 2 × 10⁴ − 18.8 ng/ml4 2 × 10³ − 11.3 ng/ml 1 0 + 5.05 ng/ml 2 2 × 10⁵ + 296. ng/ml ≈ 300ng/24 h/ 10⁶ cells 3 2 × 10⁴ + 48.0 ng/ml 4 2 × 10³ + 13.2 ng/ml

Example 3 Construction of rAAV Vectors

[0156] A. Construction of pD10-bFGF-2

[0157] The pD10 AAV vector is constructed by replacing the AAV geneencoding sequences of pD-10 (see Wang, X. et al. J. Virol. 71:3077(1997), with the CMV promoter, multiple cloning site, and BGHpolyadenylation sequences from pKm201CMV. Briefly, tides 5′-ggtatttaaaacttgcggcc gcggaatttc gactctaggc c-3′ (SEQ I.D. No. ______) and5′-gctgcccggg acttgctagc tggatgatcc tccagcgcgg ggatctcatg-3′(SEQ I.D.No. ______) are used to amplify the CMV expression cassette from pKm201CMV. The product of this PCR amplification is digested with SmaI andDraI and cloned into pD-10 digested with is new vector is namedpD10-CMV.

[0158] To construct pD10-bFGF-2, the synthetic gene for bovine FGF-2(see U.S. Pat. No. 5,464,774 for sequence) is digested with EcoRI andSalI, treated with T4 polymerase to blunt the ends, and then cloned intothe StuI site of pD10-CMV. The synthetic gene described above encodesthe mature, processed form of bovine FGF-2.

[0159] B. Construction of a rAAV Vector Expressing FGF-2-Sc.

[0160] pD10-K-FGF -2-Sc (see FIG. 31) was constructed by cloning theFGF-2 humanized bovine cDNA obtained from Scios, into the pD10 vectorbackbone containing the kanamycin (Kan) resistance gene. This cDNAcontains the mutations at amino acid positions 121 and 137 described inexample 1. Briefly, the FGF-2-Sc cDNA was digested from the parentplasmid with the enzyme NdeI, the ends blunted with T4 DNA polymerase,cut with the restriction enzyme HindIII, and cloned into the pD10-CMVvector which had been digested with the enzymes StuI and HindIII. Thenucleotide sequence of pD 1 0-K-FGF-2-Sc is illustrated in FIG. 32.

[0161] C. Infection of Cells with rAAV-FGF-2-Sc Results in theExpression of FGF-2 293 cells were infected as in example 2, with thefollowing modifications: 4×10e5 cells/well were plated in a 12 welldish, and all wells contained 3 uM etoposide. Three different particlenumbers of FGF-2 virus, and a negative control CMV-lacZ virus were usedto infect the cells. 48 hours after infection, tissue culture media wascollected and cells lysed in 100 ul lysis buffer containing Triton-X 100and Complete™ protein inhibitor cocktail (Boehringer Mannheim, Germany).FGF-2 protein in the media and lysates was assayed by ELISA. The resultsare shown below in Table 2 below. TABLE 2. TABLE 2 FGF-2 PRODUCTION IN293 CELLS FOLLOWING INFECTION WITH RAAV D10-K-FGF-2-Sc Viral Culturemedia Vector Particles Lysates (pg/mL) (pg/mL) D10-K-FGF-2-Sc 1 × 10e10324492.919 438.621 D10-K-FGF-2-Sc 1 × 10e9 32876.106 46.984D10-K-FGF-2-Sc 1 × 10e8 6950.039 14.649 CMV-lacz 1 × 10e10 2327.52717.096

Example 4 Construction of rAAV Vectors Which Express FGF5 and FGF18

[0162] A. Cloning FGF-5 into the PD10-CMV rAAV Vector.

[0163] The FGF-5 coding region (see U.S. Serial No. 08,602,147) wascloned into the rAAV pD10-CMV vector by digestion with the enzymes SacIIand Xmnl, resulting in an 814 bp fragment. This removed an ORF (ORF-1)upstream of and overlapping with the FGF-5 coding region. The ends ofthe FGF-5 fragment were then blunted with T4 DNA polymerase, and it wascloned into the rAAV pD10-CMV vector linearized with StuI. This vectorcontains a 1353 bp insertion of a bacteriophage Phi X174 HaeIIIfragment.

[0164] The pD10-CMV-FGF-5 vector is illustrated schematically in FIG. 3.In summary, this plasmid contains the CMV immediate/earlyenhancer+promoter, the CMV intron A, an FGF-5 coding region, the bovinegrowth hormone polyA site, and AAV ITR sequences. There is a 1353 bpinsertion of PhiX 174 bacteriophage DNA cloned into the NotI sitebetween one ITR and the CMV immediate early enhancer +promoter region.

[0165] B. Packaging and Functional Analysis of FGF-5 rAAV.

[0166] rAAV virus was packaged using a triple transfection method asdescribed in Example 1. However, rather than cesium chloride equilibriumdensity gradient centrifugation, heparin sulfate column chromatographyis utilized. More specifically, a cell pellet is resuspended in TNMbuffer: 20 mM Tris pH 8.0, 150 mM NaCl, 2 mM MgCl₂. Deoxycholic Acid isadded to 0.5% to lyse the cells. 50 U/ml Benzonase is added and thelysed cells are incubated at 37 degrees to digest any nucleic acids. Thecell debris is pelleted and the supernatant is filtered through a 0.45um filter and then a 0.22 um filter. The virus is then loaded onto a 1.5ml Heparin sulfate column using the Biocad HPLC. The column is thenwashed with 20 mM Tris pH 8.0, 100 mM NaCl. The rAAV particles areeluted with a gradient formed with increasing concentrations of NaCl.The fractions under the peak are pooled and filtered through a 0.22 umfilter before overnight precipitation with 8% PEG 8000. CaCl₂ is addedto 25 mM and the purified particles are pelleted and then resuspended inHBS#2: 150 mM NaCl, 50 mM Hepes pH 7.4.

[0167] Briefly, 8×10⁸ or 8×10⁹ particles of the resulting FGF-5 viruswere used to infect 293 cells, which were simultaneously treated with 3uM etoposide to enhance viral expression levels. At 24 hourspost-infection, tissue culture media and cell lysates were harvested andanalysed by Western blotting. Briefly, protein samples were run on a4-20% tris-glycine gradient gel, and transferred to nitrocellulose bystandard procedures. After blocking with 5% milk in PBS, the membranewas incubated with an anti-human FGF-5 antibody (R and D systems, madein goat) at a dilution of 1:1,000 for one hour at room temperature.After the membrane was washed 3 times in PBS+0.05% Tween-20, it wasincubated with an anti-goat secondary antibody conjugated to peroxidase(1:5,000 dilution). The membrane was then washed and the FGF-5 proteindetected by chemiluminescence.

[0168] Results of the Western blot are shown in FIG. 4. Briefly, lane 1represents 50 ng of the 29.5 Kd recombinant FGF-5 protein (R and Dsystems). Lane 2, media from cells infected with 8×10⁹ viral particlesand treated with etoposide, shows no FGF-5 expression. Lane 3 is anuninfected cell lysate control. Lane 4 and 5 are lysates from cellsinfected with 8×108 or 8×10⁹ viral particles, respectively, and Lanes 6and 7 are lysates from cells infected with 8×10⁸ or 8×10⁹ viralparticles and treated with 3 uM etoposide. Lanes 4-7 all show positiveFGF-5 expression. Lane 8 is a negative control of lysate from uninfectedcells.

[0169] In summary, although the FGF-5 signal sequence was intact, FGF-5protein was detected in the cell lysate only.

[0170] C. Cloning FGF-5 Lacking a Signal Sequence into rAAV pD10-CMV.

[0171] Oncogenic activity is associated with the wild-type FGF-5molecule (Zhan et al., 1988; Bates et al., 1991). To improve its safety,the codons for the first 21 amino acids of FGF-5's signal sequence wereremoved by PCR amplification of the above pD10-CMV-FGF-5 plasmid withthe following primers: AGA/TAT/AAG/CTT/AC C/ATG/GGT/GAA/AAG/CGT/CTC/GCC/CCC/AAA (5′, 5FGFMUTB; SEQ I.D. No.______) andCGC/GCG/CTC/GAG/AC C/ATG/AGG/AAT/ATT/ATC/CAA/AGC/GAA/ACT (3′, 3FGF5WT;SEQ I.D. No. ______). The 5′ primer contains point mutations whichdestabilize G/C rich hairpin structures of the FGF-5 mRNA, and shouldincrease levels of gene expression. The PCR product was digested withHindIII and XhoI (restriction sites introduced by the primers), andcloned by standard methods, into the pD 10 vector digested with the sameenzymes. The pD10-CMV-FGF-5 (sig−) vector is illustrated schematicallyin FIG. 5.

[0172] In summary, the pD10-CMV-FGF-5 (sig−) plasmid contains the CMVimmediate/early enhancer+promoter, the CMV intron A, the FGF-5 codingregion with the modifications described in Example C above, the bovinegrowth hormone polyA site, and the AAV ITR sequences. There is a 1353 bpinsertion of PhiX 174 bacteriophage DNA clones into the NotI sitebetween one ITR and the CMV immediate early enhancer +promoter region.

[0173] D. Western Analysis of 293 Cells Transfected with pD10-CMV-FGF-5(sig−).

[0174] Expression of FGF-5 protein was demonstrated by transienttransfection of 293 cells with the plasmid pD10-CMV-FGF-5 (sig−), bystandard methods. After 48 hours, tissue culture media and cell lysateswere harvested. Western analysis was performed with an anti-human FGF-5antibody (R and D systems) as described above.

[0175] Results of the Western analysis are provided below in FIG. 6.Briefly, lane 1 represents 50 ng of the 29.5 Kd recombinant FGF-5protein (R and D systems). Lanes 2 and 3, showing FGF-5 expression, arecell lysates from 293 cells transfected with two different clones of thepD10-CMV-FGF-5 sig− plasmid. Lane 4 is lysate from cells transfectedwith a negative control plasmid CMV-Epo. Lanes 5, 6 and 7 representmedia from cells transfected with different clones of the pD10-CMV-FGF-5sig− plasmid, respectively, and the CMV-Epo plasmid. As is evident fromthis figure, FGF-5 protein was detected in the cell lysate only.

[0176] E. Generation of FGF-5 (signal −) rAAV.

[0177] FGF-5 (sig−) rAAV virus is packaged using the triple transfectionmethod described in more detail above.

[0178] F. Cloning FGF-18 into the pD10-CMV rAAV Vector.

[0179] The FGF-18 coding region (see U.S. Provisional Application SerialNo. 60/083,553) was cloned into the pD10-CMV vector as a PstI to EcoRVfragment, using restriction sites found in both the FGF-18 and themultiple cloning site of the pD 10-CMV vector. The vector contains a1353 bp insertion of PhiX174 bacteriophage DNA (see Example A).

[0180] A schematic illustration of pD10-CMV-FGF-18 is provided in FIG.7. Briefly, this plasmid contains the CMV immediate/earlyenhancer+promoter, the CMV intron A, the FGF-18 coding region, thebovine growth hormone polyA site, and the AAV ITR sequences. There is a1353 bp insertion of PhiX 174 bacteriophage DNA cloned into the NotIsite between one ITR and the CMV immediate early enhancer+promoterregion.

[0181] G. Analysis of 293 Cells Transfected with pD10-CMV-FGF-18Plasmid.

[0182] Expression of FGF-1 8 protein was assessed by transienttransfection of 293 cells followed by Western analysis, using standardmethods. Cell lysates and tissue culture media were harvested at 48hours post transfection. An anti-peptide FGF-18 rabbit polyclonalantibody, generated against a selected polypeptide from recombinantFGF-1 8, was used at a dilution of 1:2,500 for one hour at roomtemperature. The secondary antibody, an anti-rabbit IgG conjugated toperoxidase, was used at a dilution of 1:25,000.

[0183] Results of the Western analysis are provided in FIG. 8. Briefly,lanes 1-3 represent 1, 2 and 10 ul of tissue culture media from cellstransfected with the pD 110-CMV-FGF-18 plasmid. Lane 4 is blank. Lanes5, 6 and 7 contain 2, 10 and 20 ul of lysate from the transfected cells.Lanes 8 and 9 are negative controls; 20 ul of tissue culture media andcell lysate, respectively, from uninfected cells. Lane 10 contains apositive control; an FGF-18-maltose binding protein fusion ((MBP);predicted size =80 Kd, larger than the FGF-18 protein).

[0184] H. Packaging of the pD10CMV-FGF-18 Plasmid into rAAV Particles

[0185] FGF-1 8 rAAV virus was generated by the triple transfectionmethod.

Example 5 AAV—LacZ Injected Retina

[0186] A. Subretinal Injection of rAAV

[0187] Albino Sprague-Dawley rats were injected at 14-15 days postnatal(P14-P15). Animals were anesthetized by ketamine/xylazine injection, anda local anesthetic (proparacain HCl) was applied topically to thecornea. An aperture was made through the inferior cornea of the eye witha 28 gauge needle. Subretinal injections of 2-3μl of AAV-CMV-Lac-Z werethen made by inserting a blunt 32 gauge needle through the opening anddelivering the rAAV suspension into the subretinal space in theposterior retina. The contralateral eye was either uninjected, injectedsubretinally with PBS, or with a control rAAV containing a reportergene.

[0188] B. Staining Protocol

[0189] Cryosections of the retina were stained with Bluo-gal forb-galactosidase reaction product of lacZ. In all wild type rats tested(3), positive staining was visible in the interior of the whole eyecupupon gross examination (see FIG. 9). 100 μm thick agarose or 20 μm thickcryosections of retinas indicated that most of the b-gal positivestaining localized to the photoreceptors. There were a small number ofLacZ positive retinal ganglion cells observed.

[0190] C. Anti-b-galactosidase Immunocytochemistry

[0191] Sections from 3 wildtype and 2 transgenic rats were stained witha polyclonal antibody against b-galactosidase. These results werecomparable to the bluo-gal results, primarily demonstratingphotoreceptor-specific staining. Two out of five rats showed no positivestaining.

[0192] D. Results

[0193] Subretinal injection of 2 ul of AAV-CMV-lacZ effectivelytransduced a large number of photoreceptor and retinal pigmentepithelial (rpe) cells following a single intraocular inoculation ofAAV-CMV-lacZ into the subretinal space (SRS) of the rat eye. The lateralextent of lacZ reporter gene expression was typically ⅓ to ½ of theretinal expanse following a single AAV-CMV-LacZ injection. This findingwas confirmed by bluo-gal staining of the b-galactosidase reactionproduct of the lacZ gene as well as by immunocytochemistry using anantibody specific for b-galactosidase. The AAV-CMV-lacZ vector waseffective at transducing photoreceptor and RPE cells in both the normal(wildtype) and affected, degenerating (transgenic) rat retina.

Example 6

[0194] Retinal Tissue Analysis OF rAAV-FGF-2 infected cells, vs.Controls

[0195] A. Subretinal Injection of rAAV

[0196] Line 3 albino transgenic rats (P23H-3) on an albinoSprague-Dawley background (produced by Chrysalis DNX TransgenicSciences, Princeton, N.J.) were injected at the ages of P14 or P15.Animals were anesthetized by ketamine/xylazine injection, and a localanesthetic (proparacain HCl) was applied topically to the cornea. Anaperture was made through the inferior cornea of the eye with a 28 gaugeneedle. The subretinal injections of 2 μl were then made by inserting ablunt 32 gauge needle through the opening and delivering the rAAVsuspension into the subretinal space in the posterior retina. The intentwas to inject into the subretinal space of the posterior superiorhemisphere, but sometimes upon histological examination it was foundthat the injection site was located just inferior to the optic nervehead. The opposite eye was either uninjected, injected subretinally withPBS, with control rAAV containing no neurotrophin or containing proteinsnot known to possess neurotrophic properties.

[0197] B. Histopathology Protocol/Retinal Tissue Analysis

[0198] The rats were euthanized by overdose of carbon dioxide inhalationand immediately perfused intracardially with a mixture of mixedaldehydes (2% formaldehyde and 2.5% glutaraldehyde). Eyes were removedand embedded in epoxy resin, and 1 μm thick histological sections weremade along the vertical meridian. Tissue sections were aligned so thatthe ROS and Muller cell processes crossing the inner plexiform layerwere continuous throughout the plane of section to assure that thesections were not oblique, and the thickness of the ONL and lengths ofRIS and ROS were measured as described (see e.g., LaVail, et al. PNAS1992, 89(23:11249-53 and LaVail et al., Invest. Ophthamlmol. Vis. Sci1998, 39(3):592-602). Briefly, 54 measurements of each layer orstructure were made at set points around the entire retinal section.These data were either averaged to provide a single value for theretina, or plotted as a distribution of thickness or length across theretina. The 3 greatest contiguous values for ONL thickness was alsocompared in each retina, to determine if any region of retina (e.g.,nearest the injection site) showed proportionally greater rescue;although most of these values were slightly greater than the overallmean of all 54 values, they were no different from control values thanthe overall mean. Thus, the overall mean was used in the data cited,since it was based on a much larger number of measurements.

[0199] C. Results

[0200] Two surgical methods of delivery of rAAV-CMV-FGF2 were completed,intravitreal and subretinal injection.

[0201] 1. Intravitreal injection

[0202] RAAV-CMV-FGF-2 was injected into the right eye of nine transgenicS334ter rats after day P15 (the left eye was not injected). S334(4)transgenic animals were used to assess the rescue effect ofrAAV-CMV-FGF-2 on degenerating photoreceptor cells when delivered byintravitreal injection. The rats were all sacrificed at age p60 and theembedded in plastic and sectioned to assess morphology and therapeuticeffect as assayed by the preservation of thickness of outer nuclearlayer. Superior and inferior regions of eyecup are quantitated bymeasuring the ONL thickness using a BioQuant morphometric measuringsystem (BioQuant). Injected eyes were evaluated along with uninjectedcontrol eyes.

[0203] Control Left superior—16.52+/−2.77 urn

[0204] Injected Right superior-19.71+/−5.27 urn

[0205] Control Left inferior -22.64+/−2.11 um

[0206] Injected Right inferior -26.47+/−3.55 um

[0207] Based upon these results it was evident that there is a rescueeffect of AAV -CMV-FGF-2 when delivered intraocularly into the vitreouscavity.

[0208] 2. Subretinal Injection of rAAV-CMV-FGF-2

[0209] Experiment A.—Location of injection—subretinal, 7 rats-both rightand left eyes injected, 3 rats-(left eye =uninjected). Number of ratsinjected—10 rats all wild type p15 on day of injection. One rat wassacrificed every week starting at week 2. Expression of FGF-2 wasassessed, as well as any signs of inflammation or neovascularization.

[0210] Experiment B.—Location of injection—subretinal, 5 rats—right eyesinjected w/vector left eyes injected with PBS, 4 rats—right eyesinjected w/vector (left eye=uninjected). Number of rats injected—11transgenic S334(4) rats—all were p15 on day of injection. The rats weresacrificed at age p60 and the embedded in plastic and sectioned toassess histopathology and number of surviving photoreceptor cells. .

[0211] Anatomic indication of therapeutic effect (photoreceptor rescue)was assessed histologically. Briefly, eyes injected with rAAV-CMV-FGF2retained significantly more photoreceptors at P60, P75 and P90 thanuninjected contralateral control eyes of the same animal. Retinasreceiving a subretinal injection of AAV-CMV-FGF2 at P14-15 retained 71%of the normal ONL thickness, compared to about 47% in the uninjectedcontrols (see FIGS. 11, 12, 13 and 14).

[0212] There was little or no rescue in PBS-injected control eyes(p>0.169 in all cases). This is consistent with previous reports thatneedle injury to the retina in young rats (P14-P15) does not rescuephotoreceptors or up-regulate bFGF mRNA expression.

[0213] 3. Subretinal injection of rAAV-CMV-FGF-2

[0214] Two to three microliters of rAAV-CMV-FGF-2 vector was injectedinto the subretinal space between the photoreceptors and the adjacentretinal pigment epithelium at P14 or P15. Rats were sacrificed and eyesexamined at time points between P60-P90. At these ages in uninjectedcontrol eyes of S334ter rats, the ONL thickness, which is an index ofphotoreceptor cell number, was reduced to about 60% of normal.

[0215] Evidence of anatomic rescue was found to be significant to thep=.005 confidence level in retinas transfected by rAAV-CMV-FGF-2 whencompared to the control AAV vectors or sham injection of PBS by ANOVA(analysis of Variance statistical measures). JMP statistical analysissoftware (Copyright (c) 1999 SAS Institute Inc. Cary, N.C., USA).

Example 7 Antibody Staining OF RAAV-FGF-2 Infected Cells

[0216] A. Injection Protocol

[0217] Albino Sprague-Dawley rats were injected with rAAV-CMV-FGF-2 atthe ages of P14 or P15 essentially as follows. Briefly, wild-typeanimals were anesthetized by ketamine/xylazine injection, and a localanesthetic (proparacain HCl) was applied topically to the cornea. Anaperture was made through the inferior cornea of the eye with a 28 gaugeneedle. The subretinal injections of 2-3 ul of rAAV-CMV-FGF-2 were thenmade by inserting a blunt 32 gauge needle through the opening anddelivering the rAAV suspension into the subretinal space in theposterior retina. The contralateral eye was either uninjected, injectedsubretinally with PBS, wild-type rAAV, or with rAAV-CMV-lacZ.

[0218] B. Staining Protocol

[0219] Fixed eyecups were embedded in OCT and cryosectioned in 20umthick sections. Sections from 10 wt rats were stained with antibody toFGF-2. (primary-anti-FGF-2 1:500 (commercial antibody purchased from R&Dsystems) (secondary-anti-goat Cy3 conjugate (Sigma, St. Louis. Mo.)

[0220] C. Results

[0221] Immunohistochemistry was used to look for expression of FGF-2 inthe eye. Two rats were examined every week starting at 3 weekspost-injection. Retinas were examined for expression of FGF-2 and alsoexamined histopathologically for signs of inflammation orneovascularization.

[0222] Results are shown in FIG. 15. Briefly, expression of FGF-2 wasfound in retinal photoreceptor cells as well as RPE cells at 35 daysfollowing inoculation with 2-3 ul of rAAV-CMV-FGF-2. Less significantexpression was noted in retinal bipolar interneurons and retinalganglion cells (RGCs) following injection into the subretinal space(SRS). No significant staining above background was observed in sectionsinjected with PBS or rAAV-CMV-lacZ vectors.

Example 8 Retinal Tissue Analysis OF rAAV-FGF-5 (SIG−) AND −18 InfectedCells, vs. Controls

[0223] A. Subretinal Injection of rAAV

[0224] Line 4 albino transgenic rats (S334ter-4) on an albinoSprague-Dawley background (produced by Chrysalis DNX TransgenicSciences, Princeton, N.J.) were injected at age P15. Animals wereanesthetized by ketamine/xylazine injection, and a local anesthetic(proparacain HCl) was applied topically to the cornea. An aperture wasmade through the inferior cornea of the eye with a 28 gauge needle. Thesubretinal injections of 2.5 μl were then made by inserting a blunt 32gauge needle through the opening and delivering the rAAV suspension intothe subretinal space in the posterior retina. The opposite eye waseither uninjected, injected subretinally with PBS, with control rAAVcontaining no neurotrophin or containing proteins not known to possessneurotrophic properties.

[0225] B. Histopathology Protocol/Retinal Tissue Analysis

[0226] The rats were euthanized by overdose of carbon dioxide inhalationand immediately perfused intracardially with a mixture of mixedaldehydes (2% formaldehyde and 2.5% glutaraldehyde). Eyes were removedand embedded in epoxy resin, and 1 μm thick histological sections weremade along the vertical meridian. Tissue sections were aligned so thatthe ROS and Müller cell processes crossing the inner plexiform layerwere continuous throughout the plane of section to assure that thesections were not oblique, and the thickness of the ONL was measured asdescribed (LaVail, et al). Briefly, 54 measurements of each layer orstructure were made at set points around the entire retinal section. The27 measurements from the inferior region and the superior region of theretina were averaged separately to give two values for each eye. Thisseparation was made because the retina degenerates at different rates inthese two regions of the S334ter-4 animal model.

[0227] C. Results

[0228] Sub-retinal injections of both rAAV-CMV-FGF-5 (sig−) andrAAV-CMV-FGF-18 were performed.

[0229] 1. Sub-Retinal Injection of rAAV-CMV-FGF-5 (sig−)

[0230] Experiment.—Location of injection—subretinal, 3 rats—right eyesinjected w/vector left eyes injected with PBS, 8 rats—right eyesinjected w/vector left eyes injected with rAAV-CMV-LacZ, 4 rats—righteyes injected w/vector (left eye=uninjected). Number of rats injected—15transgenic S334ter-4 rats—all were p15 on day of injection. The ratswere sacrificed at age p60. The retinas were embedded in plastic andsectioned to assess histopathology and number of surviving photoreceptorcells.

[0231] Anatomic indication of therapeutic effect (photoreceptor rescue)was assessed histologically. The injection of rAAV-CMV-FGF-5 (sig−)resulted in significant rescue of photoreceptors, compared to PBSinjected, rAAV-CMV-LacZ injected, and uninjected eyes (see FIG. 33). Therescue was significant to the p=0.05 confidence level for all threecomparisons, by ANOVA (analysis of Variance statistical measures). JMPstatistical analysis software (Copyright (c) 1999 SAS Institute Inc.Cary, N.C., USA).

[0232] 2. Sub-Retinal Injection of rAAV-CMV-FGF-18

[0233] Experiment.—Location of injection—subretinal, 3 rats—right eyesinjected w/vector left eyes injected with PBS, 3 rats—right eyesinjected w/vector left eyes injected with rAAV-CMV-LacZ, 4 rats—righteyes injected w/vector (left eye=uninjected). Number of rats injected—10transgenic S334ter-4 rats —all were p15 on day of injection. The ratswere sacrificed at p60, and the retinas embedded in plastic andsectioned.

[0234] Eyes injected with rAAV-CMV-FGF-18 retained significantly morephotoreceptors at P60 than PBS injected, rAAV-CM-LacZ injected, oruninjected control eyes. Each comparison, by ANOVA, was statisticallysignificant to the p=0.05 confidence level.

Example 9 In Vivo Delivery to Retinal Ganglion Cells (RGCS)

[0235] A. Intra-Vitreal Injection of AAV Vectors

[0236] All surgical procedures were performed in female adultSprague-Dawley rats (180-200 g; Charles River Breeders) under generalanesthesia (7% chloral hydrate; 0.42 mg per g of body weight, i.p.) inaccordance with the Use of Animals in Neuroscience Research and McGillUniversity Animal Care Committee guidelines for the use of experimentalanimals.

[0237] Briefly, rAAV-CMV-lacZ (5 μl; see above) was injected into thevitreous chamber in the superior (dorsal) hemisphere of the retina usinga posterior approach as described (Di Polo, PNAS, 1998). Control eyeswere injected with equal volumes of Hepes-buffered saline (HBS, virusvector).

[0238] B. Identification of RPCs

[0239] For visualization of RGCs, neurons were retrogradely labeled withthe fluorescent tracer Fluorogold (Fluorochrome, Englewood, Colo.) at 2%in 0.9% NaCl containing 10% dimethyl sulfoxide by application of thetracer to both superior colliculi 7 days prior to analyses as described(Vidal-Sanz et al., 1988). Anesthetized rats were then perfusedintracardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB,pH 7.4) and the eyes were immediately enucleated. The anterior part ofthe eye and the lens were removed and the remaining eye cup was immersedin the same fixative for 2 hr at 4° C. Eye cups were cryoprotected ingraded sucrose solutions (10-30% in PB) for several hours at 4° C.,embedded in O.C.T. compound (Tissue-Tek, Miles Laboratories, Elkhart,IN) and frozen in a 2-methylbutane/liquid nitrogen bath. Retinal radialcryosections (12-15 em), obtained along the vertical meridian of theeye, were collected onto gelatin-coated slides and processed forimmunocytochemistry. Alternatively, entire eyes were rinsed three times(15 min each) in PBS at room temperature with gentle shaking, towhole-mount histochemical staining as described below.

[0240] C. Histochemical Analysis

[0241] Expression of the bacterial LacZ gene in whole retinas wasdetected by standard histochemical staining reactions using halogenatedindoyl-β-D-galactoside (Bluogal; GIBCO BRL). Following removal of theanterior eye structures and lens, eye cups were incubated overnight in astaining solution containing 5 mM K-ferricyanide, 5 mM K-ferrocyanide, 2mM MgCl₂, and 0.5 mg/ml Bluo-gal at 37° C. Retinas were then dissected,fixed for an additional 30 min and flat-mounted vitreal side up on glassslides.

[0242] For visualization of the AAV-mediated lacZ gene product inretinal radial sections, cryosections were incubated in 10% normal goatserum (NGS) in 0.2% Triton X-100 (Sigma, St. Louis, Mo.) in phosphatebuffer saline (PBS) for 30 min at room temperature to block non-specificbinding. Two primary antibodies raised against the lacZ gene productwere used with similar results. A polyclonal anti-betagal antibody(diluted 1:1000; 5 prime→3 prime, Inc., Boulder, Colo.) and a monoclonalanti-LacZ antibody (diluted 1:500; Promega, Madison, Wis.). Primaryantibodies were added in 2% NGS in 0.2% Triton X-100 and incubatedovernight at 4° C. Sections were subsequently processed with anti-rabbitCy3-conjugated IgG (diluted 1:500, Jackson Immunoresearch, West Grove,Pa.) or anti-mouse Cy3-conjugated IgG (diluted 1:500, JacksonImmunoresearch) and mounted. Control sections were treated in the sameway but with omission of the primary antibody. Sections were visualizedby fluorescent microscopy (Polyvar, Reichert-Jung).

[0243] Expression of the heparan sulfate proteoglycan receptor in theretina was examined using a monoclonal anti-heparan sulfate antibody(HepSS-1, diluted 1:1,000, Seikagaku Corporation, Tokyo, Japan).Following overnight incubation at 4° C., sections were processed withbiotinylated anti-mouse Fab fragment (Jackson Immunoresearch),avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame,Calif.), followed by reaction in a solution containing 0.05%diaminobenzidine tetrahydrochloride (DAB) and 0.06% hydrogen peroxide inPB (pH 7.4) for 5 min. For analysis of co-localization of heparansulfate in Fluorogold-labeled neurons, sections were processed withCy3-coupled anti-mouse IgG (Jackson Immunoresearch) after incubation inprimary antibody. In all cases, the primary antibody was omitted incontrol sections. Sections were mounted and visualized by light orfluorescent microscopy.

[0244] Quantification of AAV-transduced cells in the ganglion cell layerof retinal flat-mounts was performed in two ways: i) by counting theentire number of Bluo-gal positive cells in each of the retinalquadrants (superior, inferior, temporal and nasal); and ii) by countingthe number of cells in three standard areas (at 1, 2 and 3 mm from theoptic disc) of each quadrant as previously described (Villegas-Perez etal., 1993).

[0245] For quantification of Fluorogold, Bluo-gal or HepSS-1 positivecells in retinal radial sections, the entire number of labeled cells persection was counted under fluorescent microscopy. Four to five serialsections per eye were routinely counted and a mean value per animal wasobtained, followed by the calculation of a mean value for the entireexperimental group which consisted of 4-5 rats. Results were analyzedusing the Sigmastat program (Jandel, San Rafael Madera, Calif.) by astudent's t test (paired groups).

[0246] D. Results

[0247] Analysis of retinas from eyes that received a single intravitrealinjection of rAAV-CMV-lacZ demonstrated a large number of LacZ-positivecells throughout the entire GCL as assessed by histochemical LacZstaining of both flat-mounts (FIG. 16A) and radial sections (FIG. 16B).In many cases, RGCs transduced by AAV could be unequivocally identifiedbecause the LacZ reaction product filled their axons that converged atthe level of the optic nerve head. In addition, LacZ-positivephotoreceptor nuclei were observed but were always restricted to thevicinity of the injection site (not shown). No staining was observed incontrol eyes injected with virus vector. No signs of cytotoxic damage orcellular immune reaction to the viral vector were observed in any of theretinas examined.

[0248] Quantification of LacZ-positive cells in the GCL of retinalflat-mounts demonstrated a 2.8-fold increase between 2 and 4 weeks afterintravitreal injection of the rAAV-CMV-lacZ vector (FIG. 17). Forexample, it was found that 27,569±7,646 cells/retina (mean±S.D.;n=3) and79,043±10,321 cells/retina (n=4) expressed the LacZ gene product at 2and 4 weeks after intraocular administration of the vector,respectively. A comparable number of cells expressing the AAV-mediatedtransgene at 4 weeks was observed at 8 weeks (70,221±12,500; n=3)following rAAV-CMV-lacZ injection. Although the majority ofLacZ-positive cells were observed in the superior hemisphere at 2 weeksafter virus administration, there was robust transgene expressionthroughout the entire retina by 4 and 8 weeks as assessed byquantification of Lac-Z positive cell densities in all retinal quadrantsat these time points (FIG. 17).

[0249] To identify the cellular localization of the AAV-mediated LacZgene product, immunocytochemical staining of LacZ was combined withretrograde tracing of RGC bodies using Fluorogold backlabeling from thesuperior colliculi. Double-labeling experiments indicated that themajority of cells in the GCL expressing the LacZ gene product were alsoFluorogold-positive (FIG. 18). Analysis of the number ofFluorogold-labeled cells in the GCL expressing the LacZ gene productindicated that 300±29 (mean ±S.D.; n=4) expressed both markers out of330±32 Fluorogold-labeled cells (the average RGC population per retinalradial section). This indicates that ˜92% of RGCs, identified by theFluorogold label, also expressed the AAV-mediated LacZ gene product(FIG. 19). In all retinas examined, it was routinely observed that anumber of Lac-Z positive cells that were not labeled with Fluorogold(FIGS. 18 and 19). Thus, it is possible that these cells are displacedamacrine cells or RGCs that failed to incorporate the retrograde tracer.Together, these results indicate that RGCs are preferentially transducedby recombinant AAV following intravitreal injection of this viralvector.

[0250] To investigate the molecular mechanisms underlying preferentialtransduction of RGCs by AAV, expression of the heparan sulfateproteoglycan (which mediates both AAV attachment and infection of targetcells (Summerford et al., 1998)) in the adult retina was observed.Immunostaining of retinal radial sections with a specific antibodyagainst heparan sulfate (HepSS-1) demonstrated robust staining in theGCL (FIG. 20). Positive immunolabeling was clearly visualized in bothneuronal somata and axonal bundles in the fiber layer. More diffuse andsparse staining was observed in some photoreceptor nuclei and cells inthe inner nuclear layer. No staining was observed in control retinalsections in which the primary antibody was omitted (not shown).

[0251] To determine the cell type within the GCL that express theheparan sulfate proteoglycan receptor, a double-labeling study wasperformed in which RGCs were first retrogradely labeled from thesuperior colliculi followed by immunostaining of retinal sections withHepSS-1. Our analysis showed that 299+23 (mean ±S.D.; n—4) cells in theGCL expressed both Fluorogold and HepSS-1 markers out of 315+34Fluorogold-labeled cells which represent the average RGC populationvisualized per retinal radial section. The large population of RGCs(˜95%) expressing heparan sulfate proteoglycan receptor correlated wellwith the number of RGCs expressing the AAV-mediated transgene product(˜92%). Together, these data suggest that preferential transduction ofadult RGCs by recombinant AAV is mediated by the heparan sulfateproteoglycan receptor expressed by these neurons.

Example 10 Construction of a rAAV Vector Expressing Vascular EndothelialGrowth Factor (VEGF) 165

[0252] The human VEGF-165 cDNA was cloned from the PCR-Blunt II TopoVector (Invitrogen) into the pD10-CMV rAAV vector as an EcoRI fragment(pD10 -VEGFuc). The pD10-VEGFuc vector is illustrated schematically inFIG. 21, and its nucleotide sequence is shown in FIG. 22. The VEGFucrAAV virus was packaged using the triple transfection method andpurified by column chromatography.

[0253] Briefly, a cell pellet is resuspended in TNM buffer: 20 mM TrispH 8.0, 150 mM NaCl, 2 mM MgCl₂. Deoxycholic Acid is added to 0.5% tolyse the cells. 50u/ml Benzonase is added and the lysed cells areincubated at 37 degrees to digest any nucleic acids. The cell debris ispelleted and the supernatant is filtered through a 0.45 um filter andthen a 0.22 um filter. The virus is then loaded onto a 1.5 ml Heparinsulfate column using the Biocad. The column is then washed with 20 mMTris pH 8.0, 100 mM NaCl. The rAAV particles are eluted with a gradientformed with increasing concentrations of NaCl. The fractions under thepeak are pooled and filtered through a 0.22 um filter before overnightprecipitation with 8% PEG 8000. CaCl₂ is added to 25 mM and the purifiedparticles are pelleted and then resuspended in HBS#2: 150 mM NaCl, 50 mMHepes pH 7.4.

Example 11 Infection of 293 Cells with D10-VEGF rAAV Results in VEGFProtein Expression

[0254] The functionality of the viral particles was assessed byinfection of 293 cells with 3 different viral multiplicities ofinfection (MOIs); 1×10e7, 1×10e8 and 1×10e9 viral particles per 4×10e5293 cells, in the presence of 1.5 uM etoposide. At 48 hours postinfection, tissue culture media (sups) and cell lysates were harvested.VEGF protein levels were determined using a Quantikine human VEGFsandwich ELI SA kit (R and D Systems, see FIG. 23). VEGF proteinconcentrations are given in pg/ml. The two highest MOIs gave valuessignificantly above that of the cells infected with a negative controlvirus. The levels of secreted VEGF were approximately 4-7 fold higherthan those of the lysates.

Example 12 Infection of Retinal Pigment Epithelial (RPE) Cells with D10-VEGF rAAV Results in VEGF Protein Secretion

[0255] VEGF expression levels in a monolayer of cultured primary (orvery early passage) human fetal RPE cells infected with D10-VEGF 165rAAV were clearly elevated relative to endogenous levels. Cells wereinfected with a range of rAAV particles from 0 to 1×10e5 per cell. VEGFexpression was dose dependent, increased over time, and secretionappeared to be somewhat higher from the apical surface. In arepresentative experiment, RPE cells infected with 1×10e5 viralparticles secreted >100 ng/1×10e6 cells from the apical surface and 50ng/1×10e6 cells from the basal surface at 8 days post infection. Thepolarity of VEGF secretion from human fetal RPE cells infected with 3different MOIs is shown in FIG. 24.

Example 13 Infection of Retinal Pigment Epithelial (RPE) Cells with VEGFrAV Results In VEGF Protein Secretion and Decreased Membrane Conductance

[0256] Infection of cultured human fetal RPE cells with recombinant VEGFadenovirus (AV) results in secretion of very high levels of VEGF fromboth the apical and basal surfaces of the RPE. MOI's of 0 to 1,000 or 0to 10,000 particles per cell were used in two separate experiments. Inboth cases, expression levels increased over time, peaking atapproximately 100-200 ug/1×10e6 cells at the highest MOI's (see FIG.25). In addition, the total transepithelial membrane resistance of theRPE monolayer decreased significantly at all MOIs, and by approximately4-5 fold at the highest MOIs (see FIG. 26).

Example 14 Construction of a rAAV Vector Expressing Soluble FLT-1(SFLT-1) Receptor

[0257] The sFlt-1 cDNA was cloned from the Blunt II Topo Vector(Invitrogen) into the pD10-CMV rAAV vector as an EcoRI fragment(pD10-sFlt-1). The pD10-sFlt-1 vector is illustrated schematically inFIG. 27, and its nucleotide sequence is shown in FIG. 28. The humansFlt-1 rAAV virus was packaged using the triple transfection techniqueand purified by column chromatography.

Example 15 In Vitro Assay for Anti-Angiogenic Activity

[0258] This example describes the HMVEC (human dermal microvascularendothelial cell) proliferation assay, which can be utilized todetermine the anti-angiogenic activity of a molecule by inhibition ofVEGF stimulated proliferation (see Kupprion et al., 1998. JBC273:29635-29640).

[0259] Briefly, HMVEC cells obtained from Clonetics (catalog #2543), areseeded in a 96 well culture dish at a density of 2,000 cells per well in100 ul assay media and incubated at 37° C. for 3-5 hours. The assaymedia is EBM media, or endothelial basal media (Clonetics, catalog#CC-3121) containing 5% FBS and 1% pen/strep. Dilutions ofanti-angiogenic samples are added in triplicate (50 ul each, for finalwell volume of 200 ul), immediately followed by 50 ul 20 ng/mlrecombinant VEGF (R and D Systems, final concentration 5 ng/ml, or 0.1nM). The samples are conditioned media from 293 cells transientlytransfected with a pD 10 rAAV plasmid, by standard methods, or from 293cells infected with an rAAV virus. Culture media is collected from 24-48hours post-transfection or post-infection. After the addition of sampleand recombinant VEGF, the cells are incubated for 48 hours at 37° C.,when they are pulsed with 1 uCi/well H-thymidine (Amersham, catalog#TRK300). The stock of 1 mCi/ml ³H-thymidine is diluted 1:10 in assaymedia, and 10 ul added per well. The cells are incubated for anadditional 18 hours, at which point 100 ul media is removed and thecells are lysed by the addition of 40 ul of 1M NaOH per well. Finally,the cells are harvested with a Tomtec Harvestor, transferred toFiltermat paper (Wallac), 10 ml of scintillation fluid added and theincorporation of ³H-thymidine determined by scintillation counting.

[0260] Inhibition of HMVEC proliferation by conditioned media fromsFlt-1 rAAV infected 293 cells is shown in FIG. 34. Complete inhibitionof proliferation (induced by 0.1 nM VEGF) was observed at approximatelyat 1 nM sFlt-1, and partial inhibition at approximately 0.3 nM and 0.1nM sFlt-1 protein. This inhibition was not seen with HMVEC cells treatedwith conditioned media from lacZ rAAV infected cells, or cells treatedwith recombinant VEGF alone. To generate conditioned media, 4×10e5 293cells were infected with 1×10e11total particles sFlt-1 rAAV or aCMV-lacZ control rAAV. If one assumes an infectious particle ratio of1:1,000 to 1:10,000, this is an MOI of less than or equal to 2.5×10e2per cell. All samples were run in triplicate, and means and standarddeviations are shown. Background incorporation of ³H-thymidine in cellsnot stimulated with exogenous VEGF was subtracted from all samples,leading to a negative value in one case. The total amount of sFlt-1protein in the conditioned media was determined by a sandwich Elisa(antibodies purchased from R and D Systems).

Example 16 Animal Model for Neovascularization by Subretinal Injectionof VEGF rAAV

[0261] This example describes an animal model that, after subretinalinjection of a recombinant virus (rAAV) containing an angiogenictransgene (VEGF), generates subretinal neovascularization and choroidalneovascularization. As noted above, choroidal neovascularization is ahallmark of exudative or wet Age-related Macular Degeneration (AMD), theleading cause of blindness in the elderly population. Retinalneovascularization occurs in diseases such as diabetic retinopathy andretinopathy of prematurity (ROP), the most common cause of blindness inthe young.

[0262] Briefly, subretinal injections of 2 μl AAV-VEGF (titer:5.8×0.1013 particles/ml) were made in a bleb under the retina justoutside the apical membrane of the RPE. These injections were made 3-3.5months before the animals were sacrificed. After sacrifice animals wereexamined for the extent and duration of neovascularization induced byrAAV vectors using fundus photography, fluorescein angiography (FIG.35), histology (FIG. 36), and immunochemistry (FIG. 37). As described inmore detail below, these figures demonstrate in three different waysthat AAV mediated overexpression of VEGF in the RPE can generatechoroidal neovascularization.

[0263] More specifically, FIG. 35A is an image of the fundus showingretinal blood vessels from a live animal before it was sacrificed forthe serial sectioning shown in FIG. 36. Note that the blood vessels arelarger in diameter in the AAV-VEGF injected area (black arrow). FIG.35B, is a fluorescein angiogram from the same animal taken shortly afterthe fundus image in 35A. Two minutes after fluorescein injection(intramuscular) significant leakage of fluorescein was observed at theAAV-VEGF injection site (black arrows).

[0264]FIG. 36 (panels A-D) are a series of epoxy sections taken aftersacrifice of the animal. FIG. 36A is a section taken from half of theeye furthest from AAV-VEGF injection site, showing normal photoreceptorsand blood vessels (the arrow points to photoreceptors with normalmorphology). Moving from this section toward the AAV-VEGF injectionsite, monotonically increasing photoreceptor disorganization and newblood vessel formation was observed (data not shown).

[0265]FIGS. 36B and C are epoxy sections from the AAV-VEGF injectionsite. The short arrows show new blood vessel growth and the long arrowsshow a pathologically disorganized photoreceptor layer. FIG. 36D is alsoan epoxy section from the AAV-VEGF injection site. The arrow points to ablood vessel breaking through Bruch's Membrane, probably from thechoroid.

[0266] FIGS. 37(A-D) show lectin/BrdU double-staining of rat retina(A-B) and choroid (C-D). Green staining is the lectin staining ofendothelial cells, and red staining is BrdU staining of dividing cells.Green cells with red dots are dividing endothelial cells, which are partof the newly formed blood vessels.

[0267] More specifically, FIG. 37A is an image of the at the AAV-VEGFinjection site showing extensive BrdU staining. Note that there are manymore blood vessels compared to that seen at points distant from theAAV-VEGF injection site in panel B. The lectin staining is fuzzy becausethe blood vessels are bloated with dividing endothelial cells. FIG. 37Bis an image of the retina furthest from the AAV-VEGF injection site.This image shows minimal BrdU staining. In contrast, lectin staining isclear and sharply defined. FIG. 37C is an image of the choroids at theAAV-VEGF injection site, which also shows extensive BrdU staining. Thelectin image is not shown since it binds indiscriminately throughoutchoroid and sclera. FIG. 37D is an image of the choroids furthest fromthe AAV-VEGF injection site, and shows minimal BrdU staining.

Example 17 Injection of Therapeutic Anti-Angiogenic rAAV into an AnimalModel for Ocular Neovascularization

[0268] This example demonstrates the ability of anti-angiogenicmolecules to prevent neovascularization in the rat model describedabove. Briefly, in four animals rAAV-sFlt-1 or rAAV-PEDF were injectedtogether with rAAV-VEGF into the subretinal space of one eye; thecontralateral eye received an injection of rAAV-VEGF and rAAV-GFP. Sixweeks after injection, electroretinographic analysis (ERGs) wereobtained from both eyes of each animals simultaneously. The a- or b-waveamplitude from the test eye (AAV-VEGF+AAV-sFlt) is designated as A_(t)or B_(t). The a- or b-wave amplitude from the control eye(AAV-VEGF+AAV-GFP) is designated as A_(c) or B_(c), respectively. Ineach eye ERG a- and b-waves were measured and for each animal theamplitude ratio was calculated as percent change in the test eyerelative to the control eye:

ERG Amplitude Ratio=(A _(t) −A _(c))/A _(c) or (B _(t) −B _(c))/B _(c)

[0269] The amplitude ratio for a- and b-waves are plotted in FIG. 38.Briefly, significant functional rescue was obtained in three/fouranimals using sFlt-1 and two/four animals using PEDF. FIG. 39 showscomparison ERGs of test and control eye of sFlt-l treated rats. Thedark-solid line shows that the eye rescued with sFlt-1 has a- and b-waveamplitudes approximately twice as large as the control eye (light-dashedline). This data indicates that sFlt-1 or PEDF can be used to rescueretinal function that is lost during neovascularization.

[0270] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 12 1 6514 DNA Homo sapien 1 accatgtagc ggccctgcgc gctcgctcgctcactgaggc cgcccgggca aagcccgggc 60 gtcgggcgac ctttggtcgc ccggcctcagtgagcgagcg agcgcgcaga gagggagtgg 120 ccaactccat cactaggggt tccttgtagttaatgattaa cccgccatgc tacttatcta 180 cgtagccatg ctctagggaa ttggccgcggaatttcgact ctaggccatt gcatacgttg 240 tatctatatc ataatatgta catttatattggctcatgtc caatatgacc gccatgttga 300 cattgattat tgactagtta ttaatagtaatcaattacgg ggtcattagt tcatagccca 360 tatatggagt tccgcgttac ataacttacggtaaatggcc cgcctggctg accgcccaac 420 gacccccgcc cattgacgtc aataatgacgtatgttccca tagtaacgcc aatagggact 480 ttccattgac gtcaatgggt ggagtatttacggtaaactg cccacttggc agtacatcaa 540 gtgtatcata tgccaagtcc gccccctattgacgtcaatg acggtaaatg gcccgcctgg 600 cattatgccc agtacatgac cttacgggactttcctactt ggcagtacat ctacgtatta 660 gtcatcgcta ttaccatggt gatgcggttttggcagtaca ccaatgggcg tggatagcgg 720 tttgactcac ggggatttcc aagtctccaccccattgacg tcaatgggag tttgttttgg 780 caccaaaatc aacgggactt tccaaaatgtcgtaataacc ccgccccgtt gacgcaaatg 840 ggcggtaggc gtgtacggtg ggaggtctatataagcagag ctcgtttagt gaaccgtcag 900 atcgcctgga gacgccatcc acgctgttttgacctccata gaagacaccg ggaccgatcc 960 agcctccgcg gccgggaacg gtgcattggaacgcggattc cccgtgccaa gagtgacgta 1020 agtaccgcct atagactcta taggcacacccctttggctc ttatgcatgc tatactgttt 1080 ttggcttggg gcctatacac ccccgctccttatgctatag gtgatggtat agcttagcct 1140 ataggtgtgg gttattgacc attattgaccactcccctat tggtgacgat actttccatt 1200 actaatccat aacatggctc tttgccacaactatctctat tggctatatg ccaatactct 1260 gtccttcaga gactgacacg gactctgtatttttacagga tggggtccat ttattattta 1320 caaattcaca tatacaacaa cgccgtcccccgtgcccgca gtttttatta aacatagcgt 1380 gggatctccg acatctcggg tacgtgttccggacatgggc tcttctccgg tagcggcgga 1440 gcttccacat ccgagccctg gtcccatccgtccagcggct catggtcgct cggcagctcc 1500 ttgctcctaa cagtggaggc cagacttaggcacagcacaa tgcccaccac caccagtgtg 1560 ccgcacaagg ccgtggcggt agggtatgtgtctgaaaatg agctcggaga ttgggctcgc 1620 acctggacgc agatggaaga cttaaggcagcggcagaaga agatgcaggc agctgagttg 1680 ttgtattctg ataagagtca gaggtaactcccgttgcggt gctgttaacg gtggagggca 1740 gtgtagtctg agcagtactc gttgctgccgcgcgcgccac cagacataat agctgacaga 1800 ctaacagact gttcctttcc atgggtcttttctgcagtca ccgtcgtcga cctaagaatt 1860 caggcctaag cttcctaggt atcgatctcgagcaagtcta gagggagacc acaacggttt 1920 ccctctagcg ggatcaattc cgcccccccccctaacgtta ctggccgaag ccgcttggaa 1980 taaggccggt gtgcgtttgt ctatatgttattttccacca tattgccgtc ttttggcaat 2040 gtgagggccc ggaaacctgg ccctgtcttcttgacgagca ttcctagggg tctttcccct 2100 ctcgccaaag gaatgcaagg tctgttgaatgtcgtgaagg aagcagttcc tctggaagct 2160 tcttgaagac aaacaacgtc tgtagcgaccctttgcaggc agcggaaccc cccacctggc 2220 gacaggtgcc tctgcggcca aaagccacgtgtataagata cacctgcaaa ggcggcacaa 2280 ccccagtgcc acgttgtgag ttggatagttgtggaaagag tcaaatggct ctcctcaagc 2340 gtattcaaca aggggctgaa ggatgcccagaaggtacccc attgtatggg atctgatctg 2400 gggcctcggt gcacatgctt tacatgtgtttagtcgaggt taaaaaaacg tctaggcccc 2460 ccgaaccacg gggacgtggt tttcctttgaaaaacacgat aataccatgg ccgccgggag 2520 catcaccacg ctgccagccc tgccggaggacggcggcagc ggcgctttcc cgccgggcca 2580 cttcaaggac cccaagcggc tgtactgcaagaacgggggc ttcttcctgc gcatccaccc 2640 cgacggccga gtggacgggg tccgcgagaagagcgaccca cacatcaaac tacaacttca 2700 agcagaagag agaggggttg tgtctatcaaaggagtgtgt gcaaaccgtt accttgctat 2760 gaaagaagat ggaagattac tagcttctaaatgtgttaca gacgagtgtt tcttttttga 2820 acgattggag tctaataact acaatacttaccggtcaagg aaatacacca gttggtatgt 2880 ggcactgaaa cgaactgggc agtataaacttggatccaaa acaggacctg ggcagaaagc 2940 tatacttttt cttccaatgt ctgctaagagctgatcttaa tggcagcatc tgatctcatt 3000 ttacatgaag ctggtggcat ccctgtgacccctccccagt gcctctcctg gccctggaag 3060 ttgccactcc agtgcccacc agccttgtcctaataaaatt aagttgcatc attttgtctg 3120 actaggtgtc cttctataat attatggggtggaggggggt ggtatggagc aaggggcaag 3180 ttgggaagac aacctgtagg gcctgcggggtctattggga accaagctgg agtgcagtgg 3240 cacaatcttg gctcactgca atctccgcctcctgggttca agcgattctc ctgcctcagc 3300 ctcccgagtt gttgggattc caggcatgcatgaccaggct cagctaattt ttgttttttt 3360 ggtagagacg gggtttcacc atattggccaggctggtctc caactcctaa tctcaggtga 3420 tctacccacc ttggcctccc aaattgctgggattacaggc gtgaaccact gctcccttcc 3480 ctgtccttct gattttaaaa taactataccagcaggagga cgtccagaca cagcataggc 3540 tacctggcca tgcccaaccg gtgggacatttgagttgctt gcttggcact gtcctctcat 3600 gcgttgggtc cactcagtag atgcctgttgaattatcgga tccactacgc gttagagctc 3660 gctgatcagc ctcgactgtg ccttctagttgccagccatc tgttgtttgc ccctcccccg 3720 tgccttcctt gaccctggaa ggtgccactcccactgtcct ttcctaataa aatgaggaaa 3780 ttgcatcgca ttgtctgagt aggtgtcattctattctggg gggtggggtg gggcaggaca 3840 gcaaggggga ggattgggaa gacaatagcaggggggtggg cgaagaactc cagcatgaga 3900 tccccgcgct ggaggatcat ccagccaattccctagagca tggctacgta gataagtagc 3960 atggcgggtt aatcattaac tacaaggaacccctagtgat ggagttggcc actccctctc 4020 tgcgcgctcg ctcgctcact gaggccgggcgaccaaaggt cgcccgacgc ccgggctttg 4080 cccgggcggc ctcagtgagc gagcgagcgcgcagggggtg ggcgaagaac tccagcatga 4140 gatccccgcg ctggaggatc atccagccggcgtcccggaa aacgattccg aagcccaacc 4200 tttcatagaa ggcggcggtg gaatcgaaatctcgtgatgg caggttgggc gtcgcttggt 4260 cggtcatttc gaaccccaga gtcccgctcagaagaactcg tcaagaaggc gatagaaggc 4320 gatgcgctgc gaatcgggag cggcgataccgtaaagcacg aggaagcggt cagcccattc 4380 gccgccaagc tcttcagcaa tatcacgggtagccaacgct atgtcctgat agcggtccgc 4440 cacacccagc cggccacagt cgatgaatccagaaaagcgg ccattttcca ccatgatatt 4500 cggcaagcag gcatcgccat gggtcacgacgagatcctcg ccgtcgggca tgcgcgcctt 4560 gagcctggcg aacagttcgg ctggcgcgagcccctgatgc tcttcgtcca gatcatcctg 4620 atcgacaaga ccggcttcca tccgagtacgtgctcgctcg atgcgatgtt tcgcttggtg 4680 gtcgaatggg caggtagccg gatcaagcgtatgcagccgc cgcattgcat cagccatgat 4740 ggatactttc tcggcaggag caaggtgagatgacaggaga tcctgccccg gcacttcgcc 4800 caatagcagc cagtcccttc ccgcttcagtgacaacgtcg agcacagctg cgcaaggaac 4860 gcccgtcgtg gccagccacg atagccgcgctgcctcgtcc tgcagttcat tcagggcacc 4920 ggacaggtcg gtcttgacaa aaagaaccgggcgcccctgc gctgacagcc ggaacacggc 4980 ggcatcagag cagccgattg tctgttgtgcccagtcatag ccgaatagcc tctccaccca 5040 agcggccgga gaacctgcgt gcaatccatcttgttcaatc atgcgaaacg atcctcatcc 5100 tgtctcttga tcagatcttg atcccctgcgccatcagatc cttggcggca agaaagccat 5160 ccagtttact ttgcagggct tcccaaccttaccagagggc gccccagctg gcaattccgg 5220 ttcgcttgct gtccataaaa ccgcccagtctagctatcgc catgtaagcc cactgcaagc 5280 tacctgcttt ctctttgcgc ttgcgttttcccttgtccag atagcccagt agctgacatt 5340 catccggggt cagcaccgtt tctgcggactggctttctac gtgttccgct tcctttagca 5400 gcccttgcgc cctgagtgct tgcggcagcgtgaagctgtc aattccgcgt taaatttttg 5460 ttaaatcagc tcatttttta accaataggccgaaatcggc aaaatccctt ataaatcaaa 5520 agaatagccc gagatagggt tgagtgttgttccagtttgg aacaagagtc cactattaaa 5580 gaacgtggac tccaacgtca aagggcgaaaaaccgtctat cagggcgatg gcggatcagc 5640 ttatgcggtg tgaaataccg cacagatgcgtaaggagaaa ataccgcatc aggcgctctt 5700 ccgcttcctc gctcactgac tcgctgcgctcggtcgttcg gctgcggcga gcggtatcag 5760 ctcactcaaa ggcggtaata cggttatccacagaatcagg ggataacgca ggaaagaaca 5820 tgtgagcaaa aggccagcaa aaggccaggaaccgtaaaaa ggccgcgttg ctggcgtttt 5880 tccataggct ccgcccccct gacgagcatcacaaaaatcg acgctcaagt cagaggtggc 5940 gaaacccgac aggactataa agataccaggcgtttccccc tggaagctcc ctcgtgcgct 6000 ctcctgttcc gaccctgccg cttaccggatacctgtccgc ctttctccct tcgggaagcg 6060 tggcgctttc tcatagctca cgctgtaggtatctcagttc ggtgtaggtc gttcgctcca 6120 agctgggctg tgtgcacgaa ccccccgttcagcccgaccg ctgcgcctta tccggtaact 6180 atcgtcttga gtccaacccg gtaagacacgacttatcgcc actggcagca gccactggta 6240 acaggattag cagagcgagg tatgtaggcggtgctacaga gttcttgaag tggtggccta 6300 actacggcta cactagaagg acagtatttggtatctgcgc tctgctgaag ccagttacct 6360 tcggaaaaag agttggtagc tcttgatccggcaaacaaac caccgctggt agcggcggtt 6420 ttttgtttgc aagcagcaga ttacgcgcagaaaaaaagga tctcaagaag atcctttgat 6480 cttttcttac tgaacggtga tccccaccggaatt 6514 2 5610 DNA Homo sapien 2 aaaacttgcg gccgcggaat ttcgactctaggccattgca tacgttgtat ctatatcata 60 atatgtacat ttatattggc tcatgtccaatatgaccgcc atgttgacat tgattattga 120 ctagttatta atagtaatca attacggggtcattagttca tagcccatat atggagttcc 180 gcgttacata acttacggta aatggcccgcctggctgacc gcccaacgac ccccgcccat 240 tgacgtcaat aatgacgtat gttcccatagtaacgccaat agggactttc cattgacgtc 300 aatgggtgga gtatttacgg taaactgcccacttggcagt acatcaagtg tatcatatgc 360 caagtccgcc ccctattgac gtcaatgacggtaaatggcc cgcctggcat tatgcccagt 420 acatgacctt acgggacttt cctacttggcagtacatcta cgtattagtc atcgctatta 480 ccatggtgat gcggttttgg cagtacaccaatgggcgtgg atagcggttt gactcacggg 540 gatttccaag tctccacccc attgacgtcaatgggagttt gttttggcac caaaatcaac 600 gggactttcc aaaatgtcgt aataaccccgccccgttgac gcaaatgggc ggtaggcgtg 660 tacggtggga ggtctatata agcagagctcgtttagtgaa ccgtcagatc gcctggagac 720 gccatccacg ctgttttgac ctccatagaagacaccggga ccgatccagc ctccgcggcc 780 gggaacggtg cattggaacg cggattccccgtgccaagag tgacgtaagt accgcctata 840 gactctatag gcacacccct ttggctcttatgcatgctat actgtttttg gcttggggcc 900 tatacacccc cgctccttat gctataggtgatggtatagc ttagcctata ggtgtgggtt 960 attgaccatt attgaccact cccctattggtgacgatact ttccattact aatccataac 1020 atggctcttt gccacaacta tctctattggctatatgcca atactctgtc cttcagagac 1080 tgacacggac tctgtatttt tacaggatggggtccattta ttatttacaa attcacatat 1140 acaacaacgc cgtcccccgt gcccgcagtttttattaaac atagcgtggg atctccgaca 1200 tctcgggtac gtgttccgga catgggctcttctccggtag cggcggagct tccacatccg 1260 agccctggtc ccatccgtcc agcggctcatggtcgctcgg cagctccttg ctcctaacag 1320 tggaggccag acttaggcac agcacaatgcccaccaccac cagtgtgccg cacaaggccg 1380 tggcggtagg gtatgtgtct gaaaatgagctcggagattg ggctcgcacc tggacgcaga 1440 tggaagactt aaggcagcgg cagaagaagatgcaggcagc tgagttgttg tattctgata 1500 agagtcagag gtaactcccg ttgcggtgctgttaacggtg gagggcagtg tagtctgagc 1560 agtactcgtt gctgccgcgc gcgccaccagacataatagc tgacagacta acagactgtt 1620 cctttccatg ggtcttttct gcagtcaccgtcgtcgacct aagaattcgc ccttcgaaac 1680 catgaacttt ctgctgtctt gggtgcattggagccttgcc ttgctgctct acctccacca 1740 tgccaagtgg tcccaggctg cacccatggcagaaggagga gggcagaatc atcacgaagt 1800 ggtgaagttc atggatgtct atcagcgcagctactgccat ccaatcgaga ccctggtgga 1860 catcttccag gagtaccctg atgagatcgagtacatcttc aagccatcct gtgtgcccct 1920 gatgcgatgc gggggctgct gcaatgacgagggcctggag tgtgtgccca ctgaggagtc 1980 caacatcacc atgcagatta tgcggatcaaacctcaccaa ggccagcaca taggagagat 2040 gagcttccta cagcacaaca aatgtgaatgcagaccaaag aaagatagag caagacaaga 2100 aaatccctgt gggccttgct cagagcggagaaagcatttg tttgtacaag atccgcagac 2160 gtgtaaatgt tcctgcaaaa acacagactcgcgttgcaag gcgaggcagc ttgagttaaa 2220 cgaacgtact tgcagatgtg acaagccgaggcggtgagcc gggcaggagg aaggagcctc 2280 cctcagggtt tcgggaacca gatctctcaccaggaaagac tgatacagaa agggcgaatt 2340 caggcctaag cttcctaggt atcgatctcgagcaagtcta gaaagccatg gatatcggat 2400 ccactacgcg ttagagctcg ctgatcagcctcgactgtgc cttctagttg ccagccatct 2460 gttgtttgcc cctcccccgt gccttccttgaccctggaag gtgccactcc cactgtcctt 2520 tcctaataaa atgaggaaat tgcatcgcattgtctgagta ggtgtcattc tattctgggg 2580 ggtggggtgg ggcaggacag caagggggaggattgggaag acaatagcag gggggtgggc 2640 gaagaactcc agcatgagat ccccgcgctggaggatcatc cagctagcaa gtcccatcag 2700 tgatggagtt ggccactccc tctctgcgcgctcgctcgct cactgaggcc gggcgaccaa 2760 aggtcgcccg acgcccgggc tttgcccgggcggcctcagt gagcgagcga gcgcgccagc 2820 gattctcttg tttgctccag actctcaggcaatgacctga tagcctttgt agagacctct 2880 caaaaatagc taccctctcc ggcatgaatttatcagctag aacggttgaa tatcatattg 2940 atggtgattt gactgtctcc ggcctttctcacccgtttga atctttacct acacattact 3000 caggcattgc atttaaaata tatgagggttctaaaaattt ttatccttgc gttgaaataa 3060 aggcttctcc cgcaaaagta ttacagggtcataatgtttt tggtacaacc gatttagctt 3120 tatgctctga ggctttattg cttaattttgctaattcttt gccttgcctg tatgatttat 3180 tggatgttgg aattcctgat gcggtattttctccttacgc atctgtgcgg tatttcacac 3240 cgcatatggt gcactctcag tacaatctgctctgatgccg catagttaag ccagccccga 3300 cacccgccaa cacccgctga cgcgccctgacgggcttgtc tgctcccggc atccgcttac 3360 agacaagctg tgaccgtctc cgggagctgcatgtgtcaga ggttttcacc gtcatcaccg 3420 aaacgcgcga gacgaaaggg cctcgtgatacgcctatttt tataggttaa tgtcatgata 3480 ataatggttt cttagacgtc aggtggcacttttcggggaa atgtgcgcgg aacccctatt 3540 tgtttatttt tctaaataca ttcaaatatgtatccgctca tgagacaata accctgataa 3600 atgcttcaat aatattgaaa aaggaagagtatgagtattc aacatttccg tgtcgccctt 3660 attccctttt ttgcggcatt ttgccttcctgtttttgctc acccagaaac gctggtgaaa 3720 gtaaaagatg ctgaagatca gttgggtgcacgagtgggtt acatcgaact ggatctcaac 3780 agcggtaaga tccttgagag ttttcgccccgaagaacgtt ttccaatgat gagcactttt 3840 aaagttctgc tatgtggcgc ggtattatcccgtattgacg ccgggcaaga gcaactcggt 3900 cgccgcatac actattctca gaatgacttggttgagtact caccagtcac agaaaagcat 3960 cttacggatg gcatgacagt aagagaattatgcagtgctg ccataaccat gagtgataac 4020 actgcggcca acttacttct gacaacgatcggaggaccga aggagctaac cgcttttttg 4080 cacaacatgg gggatcatgt aactcgccttgatcgttggg aaccggagct gaatgaagcc 4140 ataccaaacg acgagcgtga caccacgatgcctgtagcaa tggcaacaac gttgcgcaaa 4200 ctattaactg gcgaactact tactctagcttcccggcaac aattaataga ctggatggag 4260 gcggataaag ttgcaggacc acttctgcgctcggcccttc cggctggctg gtttattgct 4320 gataaatctg gagccggtga gcgtgggtctcgcggtatca ttgcagcact ggggccagat 4380 ggtaagccct cccgtatcgt agttatctacacgacgggga gtcaggcaac tatggatgaa 4440 cgaaatagac agatcgctga gataggtgcctcactgatta agcattggta actgtcagac 4500 caagtttact catatatact ttagattgatttaaaacttc atttttaatt taaaaggatc 4560 taggtgaaga tcctttttga taatctcatgaccaaaatcc cttaacgtga gttttcgttc 4620 cactgagcgt cagaccccgt agaaaagatcaaaggatctt cttgagatcc tttttttctg 4680 cgcgtaatct gctgcttgca aacaaaaaaaccaccgctac cagcggtggt ttgtttgccg 4740 gatcaagagc taccaactct ttttccgaaggtaactggct tcagcagagc gcagatacca 4800 aatactgtcc ttctagtgta gccgtagttaggccaccact tcaagaactc tgtagcaccg 4860 cctacatacc tcgctctgct aatcctgttaccagtggctg ctgccagtgg cgataagtcg 4920 tgtcttaccg ggttggactc aagacgatagttaccggata aggcgcagcg gtcgggctga 4980 acggggggtt cgtgcacaca gcccagcttggagcgaacga cctacaccga actgagatac 5040 ctacagcgtg agctatgaga aagcgccacgcttcccgaag ggagaaaggc ggacaggtat 5100 ccggtaagcg gcagggtcgg aacaggagagcgcacgaggg agcttccagg gggaaacgcc 5160 tggtatcttt atagtcctgt cgggtttcgccacctctgac ttgagcgtcg atttttgtga 5220 tgctcgtcag gggggcggag cctatggaaaaacgccagca acgcggcctt tttacggttc 5280 ctggcctttt gctggccttt tgctcacatgttctttcctg cgttatcccc tgattctgtg 5340 gataaccgta ttaccgcctt tgagtgagctgataccgctc gccgcagccg aacgaccgag 5400 cgcagcgagt cagtgagcga ggaagcggaagagcgcccaa tacgcaaacc gcctctcccc 5460 gcgcgttggc cgattcatta atgcagctggcgcgctcgct cgctcactga ggccgcccgg 5520 gcaaagcccg ggcgtcgggc gacctttggtcgcccggcct cagtgagcga gcgagcgcgc 5580 agagagggag tggccaactc catcactgat5610 3 7096 DNA Homo sapien 3 aaaacttgcg gccgcggaat ttcgactctaggccattgca tacgttgtat ctatatcata 60 atatgtacat ttatattggc tcatgtccaatatgaccgcc atgttgacat tgattattga 120 ctagttatta atagtaatca attacggggtcattagttca tagcccatat atggagttcc 180 gcgttacata acttacggta aatggcccgcctggctgacc gcccaacgac ccccgcccat 240 tgacgtcaat aatgacgtat gttcccatagtaacgccaat agggactttc cattgacgtc 300 aatgggtgga gtatttacgg taaactgcccacttggcagt acatcaagtg tatcatatgc 360 caagtccgcc ccctattgac gtcaatgacggtaaatggcc cgcctggcat tatgcccagt 420 acatgacctt acgggacttt cctacttggcagtacatcta cgtattagtc atcgctatta 480 ccatggtgat gcggttttgg cagtacaccaatgggcgtgg atagcggttt gactcacggg 540 gatttccaag tctccacccc attgacgtcaatgggagttt gttttggcac caaaatcaac 600 gggactttcc aaaatgtcgt aataaccccgccccgttgac gcaaatgggc ggtaggcgtg 660 tacggtggga ggtctatata agcagagctcgtttagtgaa ccgtcagatc gcctggagac 720 gccatccacg ctgttttgac ctccatagaagacaccggga ccgatccagc ctccgcggcc 780 gggaacggtg cattggaacg cggattccccgtgccaagag tgacgtaagt accgcctata 840 gactctatag gcacacccct ttggctcttatgcatgctat actgtttttg gcttggggcc 900 tatacacccc cgctccttat gctataggtgatggtatagc ttagcctata ggtgtgggtt 960 attgaccatt attgaccact cccctattggtgacgatact ttccattact aatccataac 1020 atggctcttt gccacaacta tctctattggctatatgcca atactctgtc cttcagagac 1080 tgacacggac tctgtatttt tacaggatggggtccattta ttatttacaa attcacatat 1140 acaacaacgc cgtcccccgt gcccgcagtttttattaaac atagcgtggg atctccgaca 1200 tctcgggtac gtgttccgga catgggctcttctccggtag cggcggagct tccacatccg 1260 agccctggtc ccatccgtcc agcggctcatggtcgctcgg cagctccttg ctcctaacag 1320 tggaggccag acttaggcac agcacaatgcccaccaccac cagtgtgccg cacaaggccg 1380 tggcggtagg gtatgtgtct gaaaatgagctcggagattg ggctcgcacc tggacgcaga 1440 tggaagactt aaggcagcgg cagaagaagatgcaggcagc tgagttgttg tattctgata 1500 agagtcagag gtaactcccg ttgcggtgctgttaacggtg gagggcagtg tagtctgagc 1560 agtactcgtt gctgccgcgc gcgccaccagacataatagc tgacagacta acagactgtt 1620 cctttccatg ggtcttttct gcagtcaccgtcgtcgacct aagaattcgc cctttcacca 1680 tggtcagcta ctgggacacc ggggtcctgctgtgcgcgct gctcagctgt ctgcttctca 1740 caggatctag ttcaggttca aaattaaaagatcctgaact gagtttaaaa ggcacccagc 1800 acatcatgca agcaggccag acactgcatctccaatgcag gggggaagca gcccataaat 1860 ggtctttgcc tgaaatggtg agtaaggaaagcgaaaggct gagcataact aaatctgcct 1920 gtggaagaaa tggcaaacaa ttctgcagtactttaacctt gaacacagct caagcaaacc 1980 acactggctt ctacagctgc aaatatctagctgtacctac ttcaaagaag aaggaaacag 2040 aatctgcaat ctatatattt attagtgatacaggtagacc tttcgtagag atgtacagtg 2100 aaatccccga aattatacac atgactgaaggaagggagct cgtcattccc tgccgggtta 2160 cgtcacctaa catcactgtt actttaaaaaagtttccact tgacactttg atccctgatg 2220 gaaaacgcat aatctgggac agtagaaagggcttcatcat atcaaatgca acgtacaaag 2280 aaatagggct tctgacctgt gaagcaacagtcaatgggca tttgtataag acaaactatc 2340 tcacacatcg acaaaccaat acaatcatagatgtccaaat aagcacacca cgcccagtca 2400 aattacttag aggccatact cttgtcctcaattgtactgc taccactccc ttgaacacga 2460 gagttcaaat gacctggagt taccctgatgaaaaaaataa gagagcttcc gtaaggcgac 2520 gaattgacca aagcaattcc catgccaacatattctacag tgttcttact attgacaaaa 2580 tgcagaacaa agacaaagga ctttatacttgtcgtgtaag gagtggacca tcattcaaat 2640 ctgttaacac ctcagtgcat atatatgataaagcattcat cactgtgaaa catcgaaaac 2700 agcaggtgct tgaaaccgta gctggcaagcggtcttaccg gctctctatg aaagtgaagg 2760 catttccctc gccggaagtt gtatggttaaaagatgggtt acctgcgact gagaaatctg 2820 ctcgctattt gactcgtggc tactcgttaattatcaagga cgtaactgaa gaggatgcag 2880 ggaattatac aatcttgctg agcataaaacagtcaaatgt gtttaaaaac ctcactgcca 2940 ctctaattgt caatgtgaaa ccccagatttacgaaaaggc cgtgtcatcg tttccagacc 3000 cggctctcta cccactgggc agcagacaaatcctgacttg taccgcatat ggtatccctc 3060 aacctacaat caagtggttc tggcacccctgtaaccataa tcattccgaa gcaaggtgtg 3120 acttttgttc caataatgaa gagtcctttatcctggatgc tgacagcaac atgggaaaca 3180 gaattgagag catcactcag cgcatggcaataatagaagg aaagaataag atggctagca 3240 ccttggttgt ggctgactct agaatttctggaatctacat ttgcatagct tccaataaag 3300 ttgggactgt gggaagaaac ataagcttttatatcacaga tgtgccaaat gggtttcatg 3360 ttaacttgga aaaaatgccg acggaaggagaggacctgaa actgtcttgc acagttaaca 3420 agttcttata cagagacgtt acttggattttactgcggac agttaataac agaacaatgc 3480 actacagtat tagcaagcaa aaaatggccatcactaagga gcactccatc actcttaatc 3540 ttaccatcat gaatgtttcc ctgcaagattcaggcaccta tgcctgcaga gccaggaatg 3600 tatacacagg ggaagaaatc ctccagaagaaagaaattac aatcagaggt gagcactgca 3660 acaaaaaggc tgttttctct cggatctccaaatttaaaag cacaaggaat gattgtacca 3720 cacaaagtaa tgtaaaacat taaaggactcattaaaaagt aacagttgtc tcatatcatc 3780 ttgatttatt gtcactgttg ctaactttcaggctcaaggg cgaattcagg cctaagcttc 3840 ctaggtatcg atctcgagca agtctagaaagccatggata tcggatccac tacgcgttag 3900 agctcgctga tcagcctcga ctgtgccttctagttgccag ccatctgttg tttgcccctc 3960 ccccgtgcct tccttgaccc tggaaggtgccactcccact gtcctttcct aataaaatga 4020 ggaaattgca tcgcattgtc tgagtaggtgtcattctatt ctggggggtg gggtggggca 4080 ggacagcaag ggggaggatt gggaagacaatagcaggggg gtgggcgaag aactccagca 4140 tgagatcccc gcgctggagg atcatccagctagcaagtcc catcagtgat ggagttggcc 4200 actccctctc tgcgcgctcg ctcgctcactgaggccgggc gaccaaaggt cgcccgacgc 4260 ccgggctttg cccgggcggc ctcagtgagcgagcgagcgc gccagcgatt ctcttgtttg 4320 ctccagactc tcaggcaatg acctgatagcctttgtagag acctctcaaa aatagctacc 4380 ctctccggca tgaatttatc agctagaacggttgaatatc atattgatgg tgatttgact 4440 gtctccggcc tttctcaccc gtttgaatctttacctacac attactcagg cattgcattt 4500 aaaatatatg agggttctaa aaatttttatccttgcgttg aaataaaggc ttctcccgca 4560 aaagtattac agggtcataa tgtttttggtacaaccgatt tagctttatg ctctgaggct 4620 ttattgctta attttgctaa ttctttgccttgcctgtatg atttattgga tgttggaatt 4680 cctgatgcgg tattttctcc ttacgcatctgtgcggtatt tcacaccgca tatggtgcac 4740 tctcagtaca atctgctctg atgccgcatagttaagccag ccccgacacc cgccaacacc 4800 cgctgacgcg ccctgacggg cttgtctgctcccggcatcc gcttacagac aagctgtgac 4860 cgtctccggg agctgcatgt gtcagaggttttcaccgtca tcaccgaaac gcgcgagacg 4920 aaagggcctc gtgatacgcc tatttttataggttaatgtc atgataataa tggtttctta 4980 gacgtcaggt ggcacttttc ggggaaatgtgcgcggaacc cctatttgtt tatttttcta 5040 aatacattca aatatgtatc cgctcatgagacaataaccc tgataaatgc ttcaataata 5100 ttgaaaaagg aagagtatga gtattcaacatttccgtgtc gcccttattc ccttttttgc 5160 ggcattttgc cttcctgttt ttgctcacccagaaacgctg gtgaaagtaa aagatgctga 5220 agatcagttg ggtgcacgag tgggttacatcgaactggat ctcaacagcg gtaagatcct 5280 tgagagtttt cgccccgaag aacgttttccaatgatgagc acttttaaag ttctgctatg 5340 tggcgcggta ttatcccgta ttgacgccgggcaagagcaa ctcggtcgcc gcatacacta 5400 ttctcagaat gacttggttg agtactcaccagtcacagaa aagcatctta cggatggcat 5460 gacagtaaga gaattatgca gtgctgccataaccatgagt gataacactg cggccaactt 5520 acttctgaca acgatcggag gaccgaaggagctaaccgct tttttgcaca acatggggga 5580 tcatgtaact cgccttgatc gttgggaaccggagctgaat gaagccatac caaacgacga 5640 gcgtgacacc acgatgcctg tagcaatggcaacaacgttg cgcaaactat taactggcga 5700 actacttact ctagcttccc ggcaacaattaatagactgg atggaggcgg ataaagttgc 5760 aggaccactt ctgcgctcgg cccttccggctggctggttt attgctgata aatctggagc 5820 cggtgagcgt gggtctcgcg gtatcattgcagcactgggg ccagatggta agccctcccg 5880 tatcgtagtt atctacacga cggggagtcaggcaactatg gatgaacgaa atagacagat 5940 cgctgagata ggtgcctcac tgattaagcattggtaactg tcagaccaag tttactcata 6000 tatactttag attgatttaa aacttcatttttaatttaaa aggatctagg tgaagatcct 6060 ttttgataat ctcatgacca aaatcccttaacgtgagttt tcgttccact gagcgtcaga 6120 ccccgtagaa aagatcaaag gatcttcttgagatcctttt tttctgcgcg taatctgctg 6180 cttgcaaaca aaaaaaccac cgctaccagcggtggtttgt ttgccggatc aagagctacc 6240 aactcttttt ccgaaggtaa ctggcttcagcagagcgcag ataccaaata ctgtccttct 6300 agtgtagccg tagttaggcc accacttcaagaactctgta gcaccgccta catacctcgc 6360 tctgctaatc ctgttaccag tggctgctgccagtggcgat aagtcgtgtc ttaccgggtt 6420 ggactcaaga cgatagttac cggataaggcgcagcggtcg ggctgaacgg ggggttcgtg 6480 cacacagccc agcttggagc gaacgacctacaccgaactg agatacctac agcgtgagct 6540 atgagaaagc gccacgcttc ccgaagggagaaaggcggac aggtatccgg taagcggcag 6600 ggtcggaaca ggagagcgca cgagggagcttccaggggga aacgcctggt atctttatag 6660 tcctgtcggg tttcgccacc tctgacttgagcgtcgattt ttgtgatgct cgtcaggggg 6720 gcggagccta tggaaaaacg ccagcaacgcggccttttta cggttcctgg ccttttgctg 6780 gccttttgct cacatgttct ttcctgcgttatcccctgat tctgtggata accgtattac 6840 cgcctttgag tgagctgata ccgctcgccgcagccgaacg accgagcgca gcgagtcagt 6900 gagcgaggaa gcggaagagc gcccaatacgcaaaccgcct ctccccgcgc gttggccgat 6960 tcattaatgc agctggcgcg ctcgctcgctcactgaggcc gcccgggcaa agcccgggcg 7020 tcgggcgacc tttggtcgcc cggcctcagtgagcgagcga gcgcgcagag agggagtggc 7080 caactccatc actgat 7096 4 636 DNAHomo sapien 4 atggctccct tagccgaagt cgggggcttt ctgggcggcc tggagggcttgggccagcag 60 gtgggttcgc atttcctgtt gcctcctgcc ggggagcggc cgccgctgctgggcgagcgc 120 aggagcgcgg cggagcggag cgcgcgcggc gggccggggg ctgcgcagctggcgcacctg 180 cacggcatcc tgcgccgccg gcagctctat tgccgcaccg gcttccacctgcagatcctg 240 cccgacggca gcgtgcaggg cacccggcag gaccacagcc tcttcggtatcttggaattc 300 atcagtgtgg cagtgggact ggtcagtatt agaggtgtgg acagtggtctctatcttgga 360 atgaatgaca aaggagaact ctatggatca gagaaactta cttccgaatgcatctttagg 420 gagcagtttg aagagaactg gtataacacc tattcatcta acatatataaacatggagac 480 actggccgca ggtattttgt ggcacttaac aaagacggaa ctccaagagatggcgccagg 540 tccaagaggc atcagaaatt tacacatttc ttacctagac cagtggatccagaaagagtt 600 ccagaattgt acaaggacct actgatgtac acttga 636 5 211 PRTHomo sapien 5 Met Ala Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly LeuGlu Gly 1 5 10 15 Leu Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro ProAla Gly Glu 20 25 30 Arg Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala GluArg Ser Ala 35 40 45 Arg Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu HisGly Ile Leu 50 55 60 Arg Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His LeuGln Ile Leu 65 70 75 80 Pro Asp Gly Ser Val Gln Gly Thr Arg Gln Asp HisSer Leu Phe Gly 85 90 95 Ile Leu Glu Phe Ile Ser Val Ala Val Gly Leu ValSer Ile Arg Gly 100 105 110 Val Asp Ser Gly Leu Tyr Leu Gly Met Asn AspLys Gly Glu Leu Tyr 115 120 125 Gly Ser Glu Lys Leu Thr Ser Glu Cys IlePhe Arg Glu Gln Phe Glu 130 135 140 Glu Asn Trp Tyr Asn Thr Tyr Ser SerAsn Ile Tyr Lys His Gly Asp 145 150 155 160 Thr Gly Arg Arg Tyr Phe ValAla Leu Asn Lys Asp Gly Thr Pro Arg 165 170 175 Asp Gly Ala Arg Ser LysArg His Gln Lys Phe Thr His Phe Leu Pro 180 185 190 Arg Pro Val Asp ProGlu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu 195 200 205 Met Tyr Thr 2106 659 DNA Homo sapien 6 gagcgcagcc ctgatggaat ggatgagatc tagagttgggaccctgggac tgtgggtccg 60 actgctgctg gctgtcttcc tgctgggggt ctaccaagcataccccatcc ctgactccag 120 ccccctcctc cagtttgggg gtcaagtccg gcagaggtacctctacacag atgacgacca 180 agacactgaa gcccacctgg agatcaggga ggatggaacagtggtaggcg cagcacaccg 240 cagtccagaa agtctcctgg agctcaaagc cttgaagccaggggtcattc aaatcctggg 300 tgtcaaagcc tctaggtttc tttgccaaca gccagatggagctctctatg gatcgcctca 360 ctttgatcct gaggcctgca gcttcagaga actgctgctggaggacggtt acaatgtgta 420 ccagtctgaa gcccatggcc tgcccctgcg tctgcctcagaaggactccc caaaccagga 480 tgcaacatcc tggggacctg tgcgcttcct gcccatgccaggcctgctcc acgagcccca 540 agaccaagca ggattcctgc ccccagagcc cccagatgtgggctcctctg accccctgag 600 catggtagag cctttacagg gccgaagccc cagctatgcgtcctgactct tcctgaatc 659 7 210 PRT Homo sapien 7 Met Glu Trp Met Arg SerArg Val Gly Thr Leu Gly Leu Trp Val Arg 1 5 10 15 Leu Leu Leu Ala ValPhe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile 20 25 30 Pro Asp Ser Ser ProLeu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 35 40 45 Tyr Leu Tyr Thr AspAsp Asp Gln Asp Thr Glu Ala His Leu Glu Ile 50 55 60 Arg Glu Asp Gly ThrVal Val Gly Ala Ala His Arg Ser Pro Glu Ser 65 70 75 80 Leu Leu Glu LeuLys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly 85 90 95 Val Lys Ala SerArg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr 100 105 110 Gly Ser ProHis Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 115 120 125 Leu GluAsp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 130 135 140 LeuArg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp 145 150 155160 Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln 165170 175 Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser180 185 190 Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro SerTyr 195 200 205 Ala Ser 210 8 5974 DNA Homo sapien 8 aaaacttgcggccgcggaat ttcgactcta ggccattgca tacgttgtat ctatatcata 60 atatgtacatttatattggc tcatgtccaa tatgaccgcc atgttgacat tgattattga 120 ctagttattaatagtaatca attacggggt cattagttca tagcccatat atggagttcc 180 gcgttacataacttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 240 tgacgtcaataatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 300 aatgggtggagtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 360 caagtccgccccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 420 acatgaccttacgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 480 ccatggtgatgcggttttgg cagtacacca atgggcgtgg atagcggttt gactcacggg 540 gatttccaagtctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 600 gggactttccaaaatgtcgt aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg 660 tacggtgggaggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 720 gccatccacgctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc 780 gggaacggtgcattggaacg cggattcccc gtgccaagag tgacgtaagt accgcctata 840 gactctataggcacacccct ttggctctta tgcatgctat actgtttttg gcttggggcc 900 tatacacccccgctccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt 960 attgaccattattgaccact cccctattgg tgacgatact ttccattact aatccataac 1020 atggctctttgccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac 1080 tgacacggactctgtatttt tacaggatgg ggtccattta ttatttacaa attcacatat 1140 acaacaacgccgtcccccgt gcccgcagtt tttattaaac atagcgtggg atctccgaca 1200 tctcgggtacgtgttccgga catgggctct tctccggtag cggcggagct tccacatccg 1260 agccctggtcccatccgtcc agcggctcat ggtcgctcgg cagctccttg ctcctaacag 1320 tggaggccagacttaggcac agcacaatgc ccaccaccac cagtgtgccg cacaaggccg 1380 tggcggtagggtatgtgtct gaaaatgagc tcggagattg ggctcgcacc tggacgcaga 1440 tggaagacttaaggcagcgg cagaagaaga tgcaggcagc tgagttgttg tattctgata 1500 agagtcagaggtaactcccg ttgcggtgct gttaacggtg gagggcagtg tagtctgagc 1560 agtactcgttgctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt 1620 cctttccatgggtcttttct gcagtcaccg tcgtcgacct aagaattcag gtatggctgc 1680 tggttctatcactaccctgc cagctctgcc agaagacggt ggttctggtg ccttcccacc 1740 aggtcacttcaaagacccaa aacgtctgta ctgcaaaaac ggtggtttct tcctgcgcat 1800 ccaccccgacggccgagtgg acggggtccg cgagaagagc gacccacaca tcaaactaca 1860 acttcaagcagaagagagag gggttgtgtc tatcaaagga gtgtgtgcaa accgttacct 1920 tgctatgaaagaagatggaa gattactagc ttctaaatgt gttacagacg agtgtttctt 1980 ttttgaacgattggagtcta ataactacaa tacttaccgg tcaaggaaat acaccagttg 2040 gtatgtggcactgaaacgaa ctgggcagta taaacttgga tccaaaacag gacctgggca 2100 gaaagctatactttttcttc caatgtctgc taagagctga tcttaatggc agcatctgat 2160 ctcattttacatgaagcttc ctaggtatcg atctcgagca agtctagaaa gccatggata 2220 tcggatccactacgcgttag agctcgctga tcagcctcga ctgtgccttc tagttgccag 2280 ccatctgttgtttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2340 gtcctttcctaataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2400 ctggggggtggggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggggg 2460 gtgggcgaagaactccagca tgagatcccc gcgctggagg atcatccagc tagcaagtcc 2520 catcagtgatggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 2580 gaccaaaggtcgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 2640 gccagcgattctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag 2700 acctctcaaaaatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc 2760 atattgatggtgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac 2820 attactcaggcattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg 2880 aaataaaggcttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt 2940 tagctttatgctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg 3000 atttattggatgttggaatt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 3060 tcacaccgcatatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 3120 ccccgacacccgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 3180 gcttacagacaagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 3240 tcaccgaaacgcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 3300 atgataataatggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 3360 cctatttgtttatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 3420 tgataaatgcttcaataatg tacccgtcaa gaaggcgata gaaggcgatg cgctgcgaat 3480 cgggagcggcgataccgtaa agcacgagga agcggtcagc ccattcgctt cagcaatatc 3540 acgggtagccaacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 3600 gaatccagaaaagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 3660 cacgacgagatcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg 3720 cgcgagcccctgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 3780 agtacgtgctcgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 3840 aagcgtatgcagccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 3900 gtgagatgacaggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 3960 ttcagtgacaacgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 4020 ccgcgctgcctcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 4080 aaccgggcgcccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 4140 ttgtgcccagtcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 4200 tccatcttgttcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc 4260 cctgcgccatcagatccttg gcggcaagaa agccatccag tttactttgc agggcttccc 4320 aaccttaccagagggcgccc cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc 4380 ccagtctagctatcgccatg taagcccact gcaagctacc tgctttctct ttgcgcttgc 4440 gttttcccttgtccagatag cccagtagct gacattcatc cggggtcagc accgtttctg 4500 cggactggctttctacgtgt tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg 4560 gcagcgtgaagctgtcaatt ccgcgttaaa tttttgttaa atcagctcat tttttaacca 4620 ataggccgaaatcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag 4680 tgttgttccagtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 4740 gcgaaaaaccgtctatcagg gcgatggcgg atcagcttat gcggtgtgaa ataccgcaca 4800 gatgcgtaaggagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 4860 tgcgctcggtcgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4920 tatccacagaatcaggggat aacgcaggaa agaacatgcg gcgcgccaca tgtgagcaaa 4980 aggccagcaaaaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5040 ccgcccccctgacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5100 aggactataaagataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5160 gaccctgccgcttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 5220 tcatagctcacgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 5280 tgtgcacgaaccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 5340 gtccaacccggtaagacacg acttatcgcc actggcagca gccactggta acaggattag 5400 cagagcgaggtatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5460 cactagaaggacagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5520 agttggtagctcttgatccg gcaaacaaac caccgctggt agcggcggtt ttttgtttgc 5580 aagcagcagattacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttcttac 5640 tgaacggtgatccccaccgg aattgcggcc catgttcttt cctgcgttat cccctgattc 5700 tgtggataaccgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5760 cgagcgcagcgagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 5820 ccccgcgcgttggccgattc attaatgcag ctggcgcgct cgctcgctca ctgaggccgc 5880 ccgggcaaagcccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc 5940 gcgcagagagggagtggcca actccatcac tgat 5974 9 41 DNA Artificial SequenceOligonucleotide used for PCR amplification 9 ggtatttaaa acttgcggccgcggaatttc gactctaggc c 41 10 50 DNA Artificial Sequence Oligonucleotideused for PCR amplification 10 gctgcccggg acttgctagc tggatgatcctccagcgcgg ggatctcatg 50 11 42 DNA Artificial Sequence PCR primer 11agatataagc ttaccatggg tgaaaagcgt ctcgccccca aa 42 12 42 DNA ArtificialSequence PCR primer 12 cgcgcgctcg agaccatgag gaatattatc caaagcgaaa ct 42

We claim:
 1. A method of treating or preventing diseases of the eye,comprising, administering intraocularly a gene delivery vector whichdirects the expression of a neurotrophic factor, such that said diseaseof the eye is treated or prevented.
 2. The method according to claim 1wherein said neurotrophic factor is NGF, BDNF, CNTF, NT-3, or, NT-4. 3.The method according to claim 1 wherein said neurotrophic factor is aFGF.
 4. The method according to claim 3 wherein said FGF is FGF-2,FGF-5, FGF-18, FGF-20, or, FGF-21.
 5. The method according to claim 1wherein said disease of the eye is macular degeneration.
 6. The methodaccording to claim 1 wherein said disease of the eye is diabeticretinopathy.
 7. The method according to claim 1 wherein said disease ofthe eye is an inherited retinal degeneration.
 8. The method according toclaim 7 wherein said inherited retinal degeneration is retinitispigmentosa.
 9. The method according to claim 1 wherein said disease ofthe eye is glaucoma.
 10. The method according to claim 1 wherein saiddisease of the eye is a surgery-induced retinopathy.
 11. The methodaccording to claim 1 wherein said disease of the eye is retinaldetachment.
 12. The method according to claim 1 wherein said disease ofthe eye is a photic retinopathy.
 13. The method according to claim 1wherein said disease of the eye is a toxic retinopathy.
 14. The methodaccording to claim 1 wherein said disease of the eye is a trauma-inducedretinopathy.
 15. The method according to claim 1 wherein said genedelivery vector is a retrovirus selected from the group consisting ofHIV and FIV.
 16. The method according to claim 1 wherein said genedelivery vector is a recombinant adeno-associated viral vector.
 17. Amethod of inhibiting neovascular disease of the eye, comprising,administering intraocularly a gene delivery vector which directs theexpression of an anti-angiogenic factor, such that said neovasculardisease of the eye is inhibited.
 18. The method according to claim 17wherein said anti-angiogenic factor is soluble Flt-1, PEDF, solubleTie-2 receptor, or, a single chain anti-VEGF antibody.
 19. The methodaccording to claim 17 wherein said neovascular disease of the eye isdiabetic retinopathy, wet AMD, and retinopathy of prematurity.
 20. Themethod according to claim 17 wherein said gene delivery vector is aretrovirus selected from the group consisting of HIV and FIV.
 21. Themethod according to claim 17 wherein said gene delivery vector is arecombinant adeno-associated viral vector.
 22. A gene delivery vectorwhich directs the expression of a neurotrophic factor, or ananti-angiogenic factor.
 23. The gene delivery vector according to claim22 wherein said neurotrophic factor is NGF, BDNF, CNTF, NT-3, or, NT-4.24. The gene delivery vector according to claim 22 wherein saidneurotrophic factor is a FGF.
 25. The gene delivery vector according toclaim 22 wherein said FGF is FGF-2, FGF-5, FGF-18, FGF-20, or, FGF-21.26. The gene delivery vector according to claim 22 wherein saidanti-angiogenic factor is soluble Flt-1, PEDF, soluble Tie-2 receptor,or, a single chain anti-VEGF antibody.
 27. The gene delivery vectoraccording to claim 22 wherein said vector is generated from aretrovirus.
 28. The gene delivery vector according to claim 27 whereinsaid retrovirus is HIV or FIV.
 29. The gene delivery vector according toclaim 22 wherein said vector is generated from a recombinantadeno-associated virus.
 30. A non-human animal model ofneovascularization of the eye, comprising an animal having an angiogenictransgene in the eye.
 31. The non-human animal model according to claim30 wherein said neovascularization is retinal neovascularization. 32.The non-human animal model according to claim 30 wherein saidneovascularization is choroidal neovascularization.
 33. The non-humananimal model according to claim 30 wherein said animal is a mouse orrat.
 34. The non-human animal model according to claim 30 wherein saidangiogenic transgene encodes VEGF.
 35. The non-human animal modelaccording to claim 30 wherein said angiogenic transgene encodes anangiopoietin.
 36. A method for making a non-human animal model ofneovascularization of the eye, comprising administering to a non-humananimal a gene delivery vector which directs the expression of anangiogenic transgene.
 37. The method according to claim 36 wherein saidgene delivery vector is administered subretinally.
 38. The methodaccording to claim 36 wherein said gene delivery vector is administeredintravitreally.
 39. The method according to claim 36 wherein said genedelivery vector is rAV or rAAV.
 40. The method according to claim 36wherein said angiogenic transgene is a nucleic acid molecule whichencodes VEGF.
 41. The method according to claim 36 wherein saidangiogenic transgene is a nucleic acid molecule which encodes anangiopoietin.
 42. A method for determining the ability of ananti-angiogenic factor to inhibit neovascularization of the eye,comprising: (a) administering to an animal model according to any one ofclaims 30 to 35 an anti-angiogenic factor, and (b) determining theability of said anti-angiogenic factor to inhibit neovascularization ofthe eye.
 43. The method according to claim 42 wherein saidanti-angiogenic factor is administered subretinally.
 44. The methodaccording to claim 42 wherein said anti-angiogenic factor isadministered intravitreally.